P53 protein variants and therapeutic uses thereof

ABSTRACT

Proteins derived from the product of tumor suppressor gene p53 and having enhanced functions for therapeutical use are disclosed. The proteins advantageously have enhanced tumour suppressor and programmed cell death inducer functions, particularly in proliferative disease contexts where wild-type p53 protein is inactivated. Nucleic acids coding for such molecules, vectors containing same, and therapeutical use thereof, particularly in gene therapy, are also disclosed.

The present invention relates to proteins derived from the product ofthe tumour suppressor gene p53, having improved functions fortherapeutic use. It relates advantageously to proteins having tumoursuppressor and programmed cell death inducing functions superior to thatof the wild-type p53 protein, more particularly in pathologicalsituations of proliferation in which the wild-type p53 protein isinactivated. It also relates to the nucleic acids encoding thesemolecules, the vectors containing them and their therapeutic use,especially in gene therapy. The products of the invention areparticularly adapted to the restoration of the functions of p53 inpathological situations such as especially cancers.

The wild-type p53 protein is involved in regulating the cell cycle andin maintaining the integrity of the cell genome. This protein, whosemain function is to be an activator of the transcription of some genes,is capable, by a process not yet well defined, of blocking the cell inthe G1 phase of the cell cycle during the appearance of mutations duringthe replication of the genome, and of triggering a number of DNA repairprocesses. Furthermore, in the event of a malfunctioning of these repairprocesses or in the event of the appearance of mutation events which aretoo many to be corrected, this protein is capable of inducing thephenomenon of programmed cell death called apoptosis.

In this manner, the p53 protein acts as a tumour suppressor, byeliminating abnormally differentiated cells or cells whose genome hasbeen damaged.

This principal function of p53 depends on its function as transcriptionfactor, that is to say, in other words, on its double capacity torecognize specific sequences at the level of the genomic DNA and torecruit the general transcription machinery.

The p53 protein comprises 393 amino acids which define 5 functionaldomains (see FIG. 1):

the transcription activating domain consisting of amino acids 1 to 73and capable of binding some factors of the general transcriptionmachinery such as the TBP protein. This domain is also the seat for anumber of post-translational modifications. It is also the seat ofnumerous interactions of the p53 protein with numerous other proteinsand especially with the cellular protein mdm2 or the protein EBNA5 ofthe Epstein-Barr virus (EBV), which are capable of blocking the functionof the wild-type protein. Furthermore, this domain has amino acidsequences termed PEST for susceptibility to proteolytic degradation;

the DNA-binding domain located between amino acids 73 and 315. Theconformation of this central domain of p53 regulates the recognition ofDNA sequences specific for the p53 protein. This domain is the seat oftwo types of alterations which affect the function of the wild-typeprotein:

(i) the interaction with proteins blocking the function of p53, such asthe “large T” antigen of the SV40 virus or the E6 viral proteins of theHPV16 and HPV18 viruses which are capable of causing its degradation bythe ubiquitin system. The latter interaction can only occur in thepresence of the cellular protein E6ap (enzyme E3 of the ubiquitinilationcascade);

(ii) the point mutations which affect the function of p53, practicallyall of which are located in this region;

the nuclear localization signal consisting of amino acids 315 to 325 andessential for the correct directing of the protein in the compartmentwhere it will exert its principal function;

the oligomerization domain consisting of amino acids 325 to 355. This325 to 355 region forms a structure of the type: β sheet (326-334)-elbow(335-336)-α helix (337-355). The alterations of functions located inthis region are essentially due to the interaction of the wild-typeprotein with the various mutant forms which may lead to variable effectson the function of the wild-type protein;

the regulatory domain, consisting of amino acids 365 to 393, which isthe seat of a number of post-translational modifications(glycosylations, phosphorylations, attachment of RNA and the like) whichmodulate the function of the p53 protein in a positive or negativemanner. This domain plays an extremely important role in the modulationof the activity of the wild-type protein.

The function of the p53 protein can be disrupted in various ways:

blocking of its function by a number of factors such as for example the“large T” antigen of the SV40 virus, the EBNA5 protein of theEpstein-Barr virus, or the cellular protein mdm2;

destabilization of the protein by increasing its susceptibility toproteolysis, especially by interaction with the E6 protein of the humanpapilloma viruses HPV16 and HPV18, which promotes the entry of p53 intothe ubiguitinilation cycle. In this case, the interaction between thesetwo proteins can only occur through the prior attachment of a cellularprotein, the E6ap protein whose site of attachment is poorly known;

point mutations at the level of the p53 gene;

deletion of one or both p53 alleles.

The latter two types of modifications are found in about 50% of thevarious types of cancer. In this regard, the mutations of the p53 generecorded in cancer cells affect a very large portion of the geneencoding this protein, and they result in varying modifications of thefunction of this protein. It can, however, be noted that the greatmajority of these mutations are located in the central part of the p53protein which is known to be the region of contact with the genomicsequences specific for the p53 protein.

This explains why most of the mutants of the p53 protein have theprincipal characteristic of no longer being able to attach to the DNAsequences recognized by the wild-type protein and thus of no longerbeing able to exert their role as transcription factor. Moreover, somemutants appear to have acquired new functions such as the activation ofsome genes at the transcriptional level.

The range of these modifications is currently classified into threecategories:

the so-called weak mutants, of which the product is a nonfunctionalprotein which, in the case of a mutation on only one of the two alleles,does not affect the function of the wild-type protein encoded by theother allele. The principal representatives of this category are theH273 and W248 mutants, the latter being specific for the familialLi-Fraumeni syndrome for hypersensitivity to cancerous conditions.

the dominant-negative mutants, of which the product is a nonfunctionalprotein which, in the case of a mutation on only one of the two allelesand through interaction with the wild-type protein, is capable ofblocking the function of the latter by forming nonactive mixed oligomerswhich can no longer attach to the DNA sequences specific for thewild-type protein. The main representative of this category is the G281mutant.

the dominant-oncogenic mutants, of which the product is a protein whichis capable, on the one hand, of blocking the function of the wild-typeprotein like the mutants of the previous category and, on the otherhand, of promoting tumour development through poorly known mechanisms,thereby offering a gain in function. The principal representative ofthis category is the H175 mutant.

Taking into account its antitumour and apoptotic properties and itsinvolvement in numerous pathologies of the hyperproliferative type, thewild-type p53 gene has been used in gene and cell therapy procedures. Ithas in particular been proposed to treat certain hyperproliferativepathologies, and especially cancers, by in vivo administration of thewild-type p53 gene and by restoring the functions of p53. Theadministration may be preferably carried out by viral and especiallyadenoviral (WO 94/24297) or retroviral (WO 94/06910) vectors.

It has thus been shown that the introduction of a nucleic acid encodingthe wild-type p53 protein made it possible partially to restore a normalregulation of cell growth. However, while these results are encouraging,the effectiveness of these procedures is limited by the therapeuticefficacy of the p53 protein after transfer and expression in vivo inhyperproliferative cells. Indeed, hyperproliferative pathologicalsituations such as cancers result from the disruption of the equilibriumwhich is established in a network of negative and positive controls ofcell growth. The inactivation of the negative control exerted by thewild-type p53 protein through the appearance of a dominant-negative p53mutant from one of the two alleles, the overexpression of ap53-inactivating cellular partner such as for example mdm2, or even thepresence of a viral inactivator following an infection, constitute anunfavourable context for a therapy based on the reintroduction of awild-type p53 protein which has a high risk of also being inactivated.

It is therefore particularly important to be able to have p53 typeproteins having enhanced therapeutic properties. In particular, it wouldbe particularly advantageous to have p53 molecules which are activeconstitutively and insensitive to the inactivating effects of thedominant-negative and oncogenic mutants or of other cellular or viralproteins such as E6 from HPV18 and HPV16, MDM2, EBNA5 from EBV, and thelike, found in tumour cells.

Some modifications of the p53 protein have been described in the priorart. Thus, application WO 95/06661 describes modifications on someresidues of the homologous regions of the p53 protein, that is to say inregions 343-351, 372-380 and 381-393. However, these modifications arevery minor and do not allow the resulting products to escape themechanisms of inactivation of the p53 protein in vivo. Furthermore,these proteins do not appear to have an improved activity compared withthe wild-type p53 protein.

Hupp et al. (Cell Vol 71 (1992) 875) have described a derivative of p53comprising a deletion of the 30 C-terminal residues (p53ΔC-ter30).However, while this protein conserves a capacity to bind DNA, itsapoptotic properties have not been demonstrated. Furthermore, it is notresistant to the inactivation by the dominant-negative mutants.

Pietenpol et al. (PNAS 91 (1994) 1998) have described chimeric moleculesderived from the p53 protein, especially a protein VP16-p53(80-343)-GCN4. However, this molecule has a substantially reducedDNA-binding and transactivation capacity compared with the wild-type p53protein (40%). Moreover, it has a nonselective oligomerization region,with the risk of interacting with other cellular components and thus ofinducing a nonspecific cellular response. Moreover, its properties ofresistance to the mechanisms of inactivation are not indicated.

The present invention describes new variants of the p53 protein whichhave improved therapeutic properties. It describes, in particular,variants adapted to use in gene, especially anticancer, therapy. Thevariants of the invention are derived from the p53 protein by structuralmodification(s), conserve a p53-type activity and, expressed inhyperproliferative cells, exhibit at least an enhanced property comparedwith the p53 protein. This may be in particular the antiproliferativeand/or apoptotic activity. The variants of the invention advantageouslypossess an enhanced antiproliferative and/or apoptotic activity, or onewhich is more specific for the hyperproliferative cells or which is lesssensitive to the various alterations to which the wild-type p53 issubject.

A first subject of the invention relates more particularly to a variantof the p53 protein in which all or part of the oligomerization domain isdeleted and replaced by an artificial leucine zipper domain. Asindicated above, the p53 protein is inactivated by some mutants, andespecially the dominant-negative and oncogenic mutants, found in tumourcells. This inactivation is the result of the formation of inactivemixed oligomers between the wild-type p53 protein and the mutant, whichcan no longer attach to the specific sequences recognized by thewild-type p53 protein. The present invention now describes variants ofthe p53 protein which are resistant to the dominant-negative effect ofsome mutants, that is to say variants which are active in a cellularcontext exhibiting one or two mutated alleles, which is the case fornearly 90% of p53-dependent human cancers.

In the variants according to the invention, all or part of the naturaloligomerization domain of the protein, which does not distinguishbetween the wild-type and the mutant forms, is thus replaced by anequivalent domain having a specific oligomerization capacity. Thismodification is carried out using an optimized artificial leucine zipperin order to form a dimer. The molecules according to the invention,comprising such an artificial leucine zipper, are particularlyadvantageous because they form oligomers only with other moleculescarrying the same leucine zipper. They do not therefore form oligomerswith the dominant-negative or oncogenic mutants of the p53 protein,which are capable of inactivating them. Neither do they form oligomerswith other cellular proteins carrying oligomerization domains, which arealso capable of inactivating them or of inducing undesirable effects.They can only form homo-oligomers and therefore possess a highselectivity, ensuring a better activity in a context ofhyperproliferative pathology.

According to the present invention, the artificial leucine zipper domainis therefore dvantageously a domain not present in the natural tate,which ensures selectivity of oligomerization. Most preferably, theoligomerization domain is represented by the sequence SEQ ID No. 1.

In a preferred embodiment of the invention, the variants comprise adeletion of all or part of the oligomerization domain and of all or partof the regulatory domain. As indicated above, the oligomerization domainis located between residues 325-355 inclusive and the regulatory domainbetween residues 365-393 inclusive. This type of variant is completelyadvantageous because it lacks all or some of the effects of negativeregulation exerted via the C-terminal part (aa 365-393). These variantsconstitute potentially constitutively active proteins having anon-modulable and possibly enhanced activity. The entire regulatoryregion is advantageously eliminated. The preferred variants according tothe invention comprise a deletion of the C-terminal part of the p53protein, from residue 326 or 337 inclusive.

Examples of intermediates used for the construction of these variantsare especially:

pEC107 (75-325-lz) comprising a deletion of the C-terminal part of thep53 protein, from residue 326, substituted by an artificialoligomerization domain of sequence SEQ ID No. 1;

pEC110 (75-336-lz) comprising a deletion of the C-terminal part of thep53 protein, from residue 337, substituted by an artificialoligomerization domain of sequence SEQ ID No. 1.

According to an advantageous embodiment, in the variants of theinvention, the cysteine residue in position 182 of the p53 protein isreplaced by a histidine. This mutation makes it possible advantageouslyto increase the affinity of the variant for the specific bindingnucleotide sequences. The introduction of this additional modificationtherefore makes it possible to obtain a molecule having, in addition, anincreased transactivating potential.

Precise examples of intermediate constructs for the preparation ofvariants according to the invention combining these variousmodifications are especially:

pEC139 (75-325(H182)-lz) comprising a deletion of the C-terminal part ofthe p53 protein, from residue 326, substituted by an artificialoligomerization domain of sequence SEQ ID No. 1, and a histidine inposition 182;

pEC140 (75-336(H182)-lz) comprising a deletion of the C-terminal part ofthe p53 protein, from residue 337, substituted by an artificialoligomerization domain of sequence SEQ ID No. 1, and a histidine inposition 182.

Advantageously, in the variants according to the invention, all or partof the transactivating domain is also deleted and replaced by aheterologous transactivating domain. It was also indicated above thatthe transactivating functions of p53 are essential for its activity astumour suppressor or as inducer of apoptosis. To increase thetherapeutic potential of the variants according to the invention, it isparticularly advantageous to substitute the natural transactivatingdomain by a powerful heterologous transactivating domain. These variantsthus exhibit numerous advantages. They of course possess a hightransactivating activity. However, they are also made insensitive to theeffects of negative regulation which are exerted via the N-terminal part(aa 1-73). Indeed, this region contains the PEST sequences responsiblefor its proteolytic degradation. The substitution of this region by aheterologous transactivating domain lacking PEST sequences makes itpossible to reduce this negative regulation. These variants are alsocharacterized by the decrease, or even the suppression, of anyinteraction with the E6 protein from the human papilloma virus (HPV)which is capable of inducing their degradation. They are also lesssensitive to interactions with other cellular proteins such as MDM2 andEBNA which affect the activity of the wild-type p53 protein. Thevariants thus obtained therefore possess an enhanced stability. Theelimination of the domains sensitive to a negative regulation(regulatory and transactivating domains) leads, in a particularlyadvantageous manner, to molecules which are no longer the target ofproteins inducing their proteolysis or their inactivation.

Advantageously, in the variants of the invention, the transactivatingdomain is eliminated by deletion of residues 1 to 74. The intermediateconstructs used for producing such molecules are especially pEC107(75-325-lz), pEC110 (75-336-lz), pEC139 (75-325 (H182)-lz) and pEC140(75-336(H182)-lz).

According to a first embodiment, the heterologous transactivating domainis the transactivating domain of VP16. It consists advantageously ofresidues 411 to 490 of VP16, whose sequence is given in SEQ ID No. 2.Precise examples of variants according to the invention, combining thesevarious modifications, are especially:

pEC114 (VP16-75-325-lz) comprising a deletion of the N-terminal part ofthe p53 protein comprising residues 1-74, substituted by thetransactivating domain of VP16 of sequence SEQ ID No. 2 and a deletionof the C-terminal part of the p53 protein, from residue 326, substitutedby an artificial oligomerization domain of sequence SEQ ID No. 1. Thecomplete sequence of the variant pEC114 is represented in SEQ ID No. 25;

pEC116 (VP16-75-336-lz) comprising a deletion of the N-terminal part ofthe p53 protein comprising residues 1-74, substituted by thetransactivating domain of VP16 of sequence SEQ ID No. 2 and a deletionof the C-terminal part of the p53 protein, from residue 337, substitutedby an artificial oligomerization domain of sequence SEQ ID No. 1. Thecomplete sequence of the variant pEC116 is represented in SEQ ID No. 26;

pEC147 (VP16-75-325(H182)-lz) comprising a deletion of the N-terminalpart of the p53 protein comprising residues 1-74, substituted by thetransactivating domain of VP16 of sequence SEQ ID No. 2; a deletion ofthe C-terminal part of the p53 protein, from residue 326, substituted byan artificial oligomerization domain of sequence SEQ ID No. 1, and ahistidine in position 182;

pEC149 (VP16-75-336(H182)-lz) comprising a deletion of the N-terminalpart of the p53 protein comprising residues 1-74, substituted by thetransactivating domain of VP16 of sequence SEQ ID No. 2; a deletion ofthe C-terminal part of the p53 protein, from residue 337, substituted byan artificial oligomerization domain of sequence SEQ ID No. 1, and ahistidine in position 182.

Because of the abovementioned modifications, the variants of theinvention also have potentially enhanced “killer” properties which arethe stoppage of the cell cycle and apoptosis. The combination of themodifications mentioned, including the presence of a selectiveoligomerization domain and an improved transactivating power bysubstitution of the domain of origin and by the presence of a histidinein 182 indeed confer on the variants of the invention considerablyimproved therapeutic potentials. In addition, the variants according tothe invention make it possible to avoid the appearance of some(dominant-oncogenic) mutants. The gains in function of some mutants ofp53 are still poorly defined both at the level of their mechanisms andat the level of the domains of the p53 protein which are involved. It ishighly probable that some of these new functions will depend on thecombination with some effector cellular partners.

The elimination of the domains involved in these interactions, and whosetransforming properties have been demonstrated, within the moleculesdescribed in the present application are of the type which prevent theappearance of these gains in oncogenic functions. Thus, the mutationswhich would appear randomly during the preparation of clinical batchesof plasmids encoding the polypeptides described or during the productionof clinical batches of viral or chemical vectors encoding these samepolypeptides would not create a subpopulation of oncogenic molecules.

Moreover, because of the elimination of some domains of p53 which areessential for the attachment of some molecules which inhibit itsfunction, the variants of the invention also have a higher and morestable therapeutic activity. Finally, the existence of foreign units inthe various constructs of the invention (murine AS protein for example,artificial oligomerization domain, and the like) is capable oftriggering an immune reaction during the death of the transfected cellsand the release, into the extracellular medium, of these variousfragments, thus increasing the capacity of the immune system to combattumour cells.

According to another embodiment, the heterologous transactivating domainis a transactivating domain preferably active in the transformed cellsand not in the neighbouring healthy cells. The present invention indeedalso describes molecules whose function is exerted essentially intransformed cells and not in the neighbouring healthy cells. Although itseems that the exogenous expression of a wild-type p53 within adifferentiated cell comprising endogenous wild-type p53 has little or noeffect on viability, it is nevertheless advantageous to be able to havea protein which would be functional only within the targeted cell. Thisspecificity for the tumour cell versus the normal cell is currentlygreatly worked upon at the level of the specificity of the targeting ofthe viral vector or of the design of specific expression systems. Thepresent invention now describes derivatives of p53 in which one of thefunctional domains is switched off in the absence of a cellularactivator present essentially in the transformed cells.

Thus, another subject of the invention relates to a variant of the p53protein which is preferably active in the transformed cells, in which atleast one of the functional domains of p53 is deleted completely or inpart and is substituted by a heterologous domain which is preferablyactive in the transformed cells. Preferably, the functional domain ofp53 in question is the transactivating domain. Thus, a particularlypreferred subject of the invention relates to a variant of the p53protein which is preferably active in the transformed cells, in whichthe natural transactivating domain is deleted completely or in part andis substituted by a transactivating domain which is preferably active inthe transformed cells. Advantageously, the natural transactivatingdomain is deleted by elimination of residues 1-74 inclusive from p53.

The invention relates more particularly to variants of p53 which arefunctional specifically in the presence of an oncogenic Ras protein orof a mutant of p53. These molecules are obtained especially by replacingthe transactivating domain of the wild-type p53 protein with a proteindomain capable of specifically binding a transactivator or atransactivating complex present in a transformed cell.

The protein domain capable of specifically binding the transcriptionaltransactivator or the transcriptional transactivating complex present inthe molecules of the invention may be of various types. It may be inparticular an oligomerization domain in the case where thetransactivator or the transactivating complex targeted also comprisessuch a domain. It may also be any synthetic or natural domain known tointeract with the said transactivator or transactivating complex. It mayalso be an antibody or a fragment or derivative of an antibody directedagainst the transactivator or transactivating complex.

Advantageously, the heterologous domain consists of an antibody or anantibody fragment or derivative. The antibody fragments or derivativesare for example the fragments Fab or F(ab)′2, the regions VH or VL of anantibody or alternatively single-chain antibodies (ScFv) comprising a VHregion bound to a VL region by an arm. The construction of nucleic acidsequences encoding such antibodies modified according to the inventionhas been described for example in U.S. Pat. No. 4,946,778 or inapplications WO 94/02610, WO 94/29446.

A preferred construct according to the present invention comprises anScFv directed against a mutant of the p53 protein. These mutants appearin the transformed cells and possess a transactivating domain. Theirrecruitment by a variant according to the invention creates a chimericmolecule which is selectively active in the transformed cells.

According to another preferred mode, the ScFv is directed against atransactivating complex, that is to say a complex between a targetmolecule selectively present in the transformed cells, but lackingtranscriptional transactivator activity (for example an oncogenic ras),and a molecule carrying a transactivating domain. The latteradvantageously comprises a transactivating domain and a domain forselective binding to the said cellular molecule (for example an anti-rasScFv). The attachment of this molecule allows the formation of atranscriptional transactivating binary complex, which complex is thenrecruited by the variant of the invention.

Any other type of modification leading to this specificity of activitycan of course be used within the framework of the present invention,such as especially any transactivating domain specific for a cell type.

These selective variants advantageously comprise additionalmodifications in the C-terminal part as indicated above so as further toimprove their properties. Thus, they advantageously comprise a deletionof all or part of the oligomerization domain, which may be replaced byany heterologous oligomerization domain. This is more preferably anartificial oligomerization domain, as defined above.

Precise examples of variants according to the invention which arepreferably active in the transformed cells are especially:

ScFv.antip53*-75-325-lz, comprising a deletion of the N-terminal part ofthe p53 protein comprising residues 1-74, substituted by a proteindomain capable of specifically binding a mutant of the p53 protein whichis present in a transformed cell, and a deletion of the C-terminal partof the p53 protein, from residue 326, substituted by an artificialoligomerization domain of sequence SEQ ID No. 1;

ScFv.antip53*-75-325(H182)-lz, comprising, in addition, a His182mutation;

ScFv.antip53*-75-336-lz, comprising a deletion of the N-terminal part ofthe p53 protein comprising residues 1-74, substituted by a proteindomain capable of specifically binding a mutant of the p53 protein whichis present in a transformed cell, and a deletion of the C-terminal partof the p53 protein, from residue 337, substituted by an artificialoligomerization domain of sequence SEQ ID No. 1;

ScFv.antip53*-75-336(H182)-lz, comprising, in addition, a His182mutation;

ScFv.antip53*-75-393, comprising a deletion of the N-terminal part ofthe p53 protein comprising residues 1-74, substituted by a proteindomain capable of specifically binding a mutant of the p53 proteinpresent in a transformed cell;

ScFv.antip53*-75-393(H182), comprising, in addition, a histidine inposition 182;

ScFv.antip53*-75-367, comprising a deletion of the N-terminal part ofthe p53 protein comprising residues 1-74, substituted by a proteindomain capable of specifically binding a mutant of the p53 proteinpresent in a transformed cell; and a deletion of the C-terminal part,from residue 368;

ScFv.antip53*-75-367(H182), comprising, in addition, a histidine inposition 182;

ScFv.antip53*-75-AS, comprising a deletion of the N-terminal part of thep53 protein comprising residues 1-74, substituted by a protein domaincapable of specifically binding a mutant of the p53 protein present in atransformed cell; and a deletion of the C-terminal part, from residue367, supplemented with the 19 amino acids of sequence SEQ ID No. 3;

ScFv.antip53*-75-AS(H182), comprising, in addition, a histidine inposition 182.

In addition, the term preferably active indicates that these variantsexert their activity essentially when they are expressed in transformedcells. A residual activity may however exist in the nontransformedcells, but less than that observed in the transformed cells.

Another subject of the present invention relates to a variant of the p53protein comprising a deletion of the C-terminal part, from residue 367,fused with the sequence SEQ ID No. 3 (AS). This sequence corresponds tothe last 19 amino acids of the alternative splicing product of themurine p53 protein. This variant therefore exhibits a modification ofthe oligomerization domain based on a protein described in mice asalternative splicing variant of the wild-type protein in which the 27C-terminal amino acids are replaced by 19 different amino acids.

This variant has an affinity for the sequences specific for binding DNAwhich is potentially enhanced.

This variant advantageously comprises modifications in the N-terminalpart as indicated above so as to further improve its properties. Thus,it advantageously comprises a deletion of all or part of thetransactivating domain, which may be replaced by any heterologoustransactivating domain. It is more preferably the transactivating domainderived from the protein VP16 or a protein domain capable ofspecifically binding a transactivator or a transactivating complexpresent in a transformed cell. In addition, the residue 182 of the p53protein is advantageously replaced by a histidine.

Precise examples of this type of variants according to the invention areespecially:

ScFv.antip53*-75-AS, described above, pEC143 (VP16-75-AS), comprising adeletion of the N-terminal part of the p53 protein comprising residues1-74, substituted by the transactivating domain of VP16 of sequence SEQID No. 2; and a deletion of the C-terminal part, from residue 367,supplemented with the 19 amino acids of sequence SEQ ID No. 3. Thecomplete sequence of the variant pEC143 is represented SEQ ID No. 28;

ScFv.antip53*-75-AS(H182), described above;

pEC153 (VP16-75-AS(H182)), corresponds to pEC143 with a histidine inposition 182.

In a more general way, the invention relates to any chimeric proteincomprising a transactivating domain, a DNA-binding domain, a domain fordirecting into the nucleus and an oligomerization domain, in which thedomains for binding to DNA and for directing into the nucleus consist ofamino acids 75 to 325 of the human wild-type p53 protein (SEQ ID No. 4).The applicant has indeed shown that this region of the p53 protein,coupled with appropriate transactivating and oligomerization domains,allows the creation of p53 type molecules having particularlyadvantageous properties in terms of stability, of resistance to thenegative effects of the mutants of p53 and of sensitivity toinactivation by some cellular factors.

According to one variant, the domains for binding DNA and for directinginto the nucleus consist of amino acids 75 to 336 of the human wild-typep53 protein (SEQ ID No. 5).

The chimeric proteins according to the invention may comprise varioustypes of transactivating domains. This may be the transactivating domainof the p53 protein. Preferably, it is a heterologous transactivatingdomain, chosen for example from the transactivating domain of VP16 or aprotein domain capable of specifically binding a transactivator or atransactivating complex present in a transformed cell.

As regards the oligomerization domain, this is preferably an artificial,and therefore a specific, domain such as for example an artificialleucine zipper, in particular of sequence SEQ ID No. 1.

The chimeric proteins according to the invention may, in addition,comprise a histidine residue in position 182.

Precise examples of chimeric proteins as described in the presentapplication are especially pEC114, pEC116, pEC147 and pEC149.

The subject of the present invention is also any nucleic acid encoding avariant or a chimeric protein as defined above.

The nucleic acid according to the invention may be a ribonucleic acid(RNA) or a deoxyribonucleic acid (DNA). In addition, it may be acomplementary DNA (cDNA) which may comprise one or more introns of thep53 gene. It may be of human, animal, viral, synthetic or semisyntheticorigin. It may be obtained in various ways, and especially by chemicalsynthesis using the sequences presented in the application and forexample a nucleic acid synthesizer. It may also be obtained by thescreening of libraries by means of specific probes, especially asdescribed in the application. It may also be obtained by a combinationof techniques including the chemical modification (elongation, deletion,substitution and the like) of sequences screened from libraries. Ingeneral, the nucleic acids of the invention may be prepared according toany technique known to a person skilled in the art.

Preferably, the nucleic acid according to the invention is a cDNA or anRNA.

The nucleic acid according to the invention is advantageously chosenfrom:

(a) all or part of the sequences SED ID No. 25, 26, 27, 28, 29, 31, 32,33 and 34 or of their complementary strand,

(b) any sequence hybridizing with the (a) sequences and encoding aderivative according to the invention,

(c) variants of (a) and (b) resulting from the degeneracy of the geneticcode.

As indicated above, the applicant has now constructed new nucleic acidsequences encoding variant polypeptides of p53, having completelyremarkable antiproliferative and apoptotic properties. These nucleicacids can be used as therapeutic agents to produce, in cells,derivatives according to the invention which are capable of destroyingor of correcting cellular dysfunctions. To this end, the presentinvention also relates to any expression cassette comprising a nucleicacid as defined above, a promoter allowing its expression and a signalfor, termination of transcription. The promoter is advantageously chosenfrom promoters which are functional in mammalian, preferably human,cells. More preferably, this is a promoter allowing the expression of anucleic acid in a hyperproliferative cell (cancerous, restenosis and thelike). In this regard, various promoters can be used. This may be forexample the promoter of the p53 gene itself. It may also be regions ofdifferent origin (which are responsible for the expression of otherproteins, or which are even synthetic). It may thus be any promoter orderived sequence stimulating or repressing the transcription of a genein a specific manner or otherwise, inducible or otherwise, strong orweak. There may be mentioned in particular the promoter sequences ofeukaryotic or viral genes. For example, they may be promoter sequencesderived from the genome of the target cell. Among the eukaryoticpromoters, there may be used in particular ubiquitous promoters(promoter of the genes for HPRT, PGK, α-actin, tubulin and the like), ofpromoters of the intermediate filaments (promoter of the genes for GFAP,desmin, vimentin, neurofilaments, keratin and the like), promoters oftherapeutic genes (for example the promoter of the genes for MDR, CFTR,Factor VIII, ApoAI, and the like), tissue-specific promoters (promoterof the gene for pyruvate kinase, villin, fatty acid-binding intestinalprotein, smooth muscle a-actin and the like) or alternatively promotersresponding to a stimulus (receptor for the steroid hormones, receptorfor retinoic acid and the like). Likewise, there may be promotersequences derived from the genome of a virus, such as for example thepromoters of the adenovirus ElA and MLP genes, the CMV early promoter,or alternatively the RSV LTR promoter and the like. In addition, thesepromoter regions may be modified by addition of activation or regulatorysequences or of sequences allowing a tissue-specific or predominantexpression.

The present invention now provides new therapeutic agents which make itpossible, through their antiproliferative and/or apoptotic properties,to interfere with numerous cell dysfunctions. To this end, the nucleicacids or cassettes according to the invention may be injected as theyare at the level of the site to be treated, or incubated directly withthe cells to be destroyed or to be treated. It has indeed been describedthat naked nucleic acids could penetrate into cells without any specialvector. Nevertheless, it is preferred, within the framework of thepresent invention, to use a vector for administration which makes itpossible to improve (i) the efficiency of cell penetration, (ii) thetargeting, and (iii) the extra- and intracellular stability.

In a particularly preferred embodiment of the present invention, thenucleic acid or the cassette is incorporated into a vector. The vectorused may be of chemical origin (liposome, nanoparticle, peptide complex,lipids or cationic polymers, and the like), or viral origin (retrovirus,adenovirus, herpes virus, AAV, vaccinia virus and the like) or ofplasmid origin. The use of viral vectors rests on the naturaltransfection properties of viruses. It is thus possible to use, forexample, adenoviruses, herpes viruses, retroviruses and adeno-associatedviruses. These vectors are particularly efficient from the transfectionstandpoint. In this regard, a preferred subject according to theinvention consists in a defective recombinant retrovirus whose genomecomprises a nucleic acid as defined above. Another specific subject ofthe invention consists in a defective recombinant adenovirus whosegenome comprises a nucleic acid as defined above.

The vector according to the invention may also be a nonviral agentcapable of promoting the transfer and expression of nucleic acids ineukaryotic cells. Chemical or biochemical, synthetic or natural vectorsrepresent an advantageous alternative to natural viruses in particularfor reasons of convenience, safety and also by the absence of atheoretical limit as regards the size of the DNA to be transfected.These synthetic vectors have two principal functions, to compact thenucleic acid to be transfected and to promote its cellular attachment aswell as its passage through the plasma membrane and, where appropriate,the two nuclear membranes. To overcome the polyanionic nature of nucleicacids, the nonviral vectors all possess polycationic charges.

The nucleic acid or vector used in the present invention may beformulated for topical, oral, parenteral, intranasal, intravenous,intramuscular, subcutaneous, intraocular and transdermal administrationand the like. Preferably, the nucleic acid or vector is used in aninjectable form. It may therefore be mixed with any vehicle,pharmaceutically acceptable for an injectable formulation, especiallyfor a direct injection at the level of the site to be treated. This maybe, in particular, sterile or isotonic solutions, or dry, especiallyfreeze-dried, compositions which, upon addition, depending on the case,of sterilized water or of physiological saline, allow the preparation ofinjectable solutions. A direct injection of nucleic acid into thepatient's tumour is advantageous because it makes it possible toconcentrate the therapeutic effect at the level of the affected tissues.The doses of nucleic acid used may be adjusted according to variousparameters, and especially according to the gene, vector, mode ofadministration used, pathology in question or alternatively the desiredduration of treatment.

The invention also relates to any pharmaceutical composition comprisingat least one nucleic acid as defined above.

It also relates to any pharmaceutical composition comprising at leastone vector as defined above.

It also relates to any pharmaceutical composition comprising at leastone variant of p53 as defined above.

Because of their antiproliferative properties, the pharmaceuticalcompositions according to the invention are most particularly suitablefor the treatment of hyperproliferative disorders, such as especiallycancers and restenosis. The present invention thus provides aparticularly effective method for the destruction of cells, especiallyof hyperproliferative cells. It can be used in vivo or ex vivo. Ex vivo,it essentially consists in incubating the cells in the presence of oneor more nucleic acids (or of a vector or cassette or directly of thederivative). In vivo, it consists in administering to the organism anactive quantity of a vector (or of a cassette) according to theinvention, preferably directly at the level of the site to be treated(tumour in particular). In this regard, the subject of the invention isalso a method for destroying hyperproliferative cells comprisingbringing the said cells or some of them into contact with a nucleic acidas defined above.

The present invention is advantageously used in vivo for the destructionof hyperproliferating cells (i.e. undergoing abnormal proliferation). Itis thus applicable to the destruction of tumour cells or of the smoothmuscle cells of the vascular wall (restenosis). It is most particularlyappropriate for the treatment of cancers in which a mutant of p53 isobserved. By way of example, there may be mentioned: colonadenocarcinomas, thyroid cancers, lung carcinomas, myeloid leukaemias,colorectal cancers, breast cancers, lung cancers, gastric cancers,oesophageal cancers, B lymphomas, ovarian cancers, cancers of thebladder, glioblastomas, hepatocarcinomas, cancers of the bones, skin,pancreas or alternatively cancers of the kidney and of the prostate,oesophageal cancers, cancers of the larynx, head or neck cancers,HPV-positive anogenital cancers, EBV-positive cancers of thenasopharynx, cancers in which the cellular protein mdm2 isoverexpressed, and the like.

The variants of the invention are particularly effective for thetreatment of cancers in which the MDM2 protein is, in addition,overexpressed, as well as cancers linked to the HPV virus, such asHPV-positive anogenital cancers.

The present invention is described in greater detail in the exampleswhich follow, which should be considered as illustrative andnonlimiting.

LEGEND TO THE FIGURES

FIG. 1: Functional domains of the wild-type p53 protein. TA:transcription-activating domain; DNB: DNA-binding domain; NLS: nuclearlocalization signal; OL: oligomerization domain; REG: regulatory domain.

FIG. 2: Construction of a cDNA encoding the AS form of p53.

FIG. 3: Cloning of the cDNAs encoding the constructs pEC104, pEC106,pEC131, pEC132 and pEC133 and their variant H182.

FIG. 4: Construction of the variants pEC107, pEC110, pEC139 and pEC140by fusion with the artificial oligomerization domain.

FIG. 5: Construction of the variants pEC114, pEC116, pEC141, pEC143,pEC145, pEC147, pEC149, pEC151, pEC153 and pEC155.

FIG. 6: Recognition of specific double-stranded DNA sequences by thehybrid molecules of the invention. Gel retardation experiment:competition between HisV325 and wild-type p53. column 1: incubation inthe absence of HisV325 and wild-type p53, column 2: 30 ng wild-type p53,column 3: as 2+pAb421, column 4: 30 ng HisV325, column 5: as 4+pAb421,column 6: 30 ng HisV325+30 ng wild-type p53, column 7: as 6+pAb421,column 8: 30 ng HisV325+15 ng wild-type p53, column 9: as 8+pAb421,column 10: 30 ng HisV325+7.5 ng wild-type p53, column 11: as 10+pAb421,column 12: 30 ng HisV325+4.5 ng wild-type p53, column 13: as 12+pAb421,column 14: 30 ng HisV325+3 ng wild-type p53, column 15: as 14+pAb421

FIG. 7: Transactivating activity of the wild-type p53 protein and of theAS, V-325 and V-336 variants.

FIG. 8: Transactivating activity of the wild-type p53 protein and of theV-325, V-336 and V-343 variants.

FIG. 9: Expression of the variants of the invention in SAOS-2 cells

FIG. 10: Induction of the hdm2 and WAF1 genes in EB, EB-1 and EB-V325cells

FIG. 11: Effect of the E6 protein on the transactivating function of thep53 protein and of the variants of the invention in SAOS-2 cells.Quantity of the vectors CMV-construct=100 ng

FIG. 12: Effect of the E6 protein on the transactivating function of thep53 protein and of the variants of the invention in HeLa cells.

FIG. 13: Sensitivity of the wild-type p53 protein and of the variants ofthe invention to degradation induced by the E6 protein.

FIG. 14: Effect of the dominant-negative mutant of p53 H175 on thetransactivating function of the variants of the invention. Quantity ofthe vectors CMV-construct=100 ng

FIG. 15: Effect of the hdm2 protein on the transactivating function ofthe p53 protein and of the variants of the invention in SAOS-2 cells.Quantity of the vectors CMV-construct=100 ng

FIG. 16: Effect of the wild-type p53 protein and of the V-325 protein onthe growth of cells overexpressing the hdm2 protein.

FIG. 17: Induction of apoptosis by the wild-type p53 protein and theV-325 protein.

FIG. 18: Kinetics of induction of apoptosis in EB, EB-1 and EB-V325cells

EXAMPLES Example A

Construction of Various Nucleotide Fragments Necessary for Preparing theGenes Encoding the Variants of the p53 Protein

A1. Construction of the cDNA Encoding the Human Wild-type p53.

The human p53 gene was cloned by polymerase chain reaction (PCR) on DNAfrom a human placenta library (Clontech) using the oligonucleotides 5′-1and 3′-393.

Oligonucleotide 5′-1 (SEQ ID No. 6): ATGGAGGAGCCGCAG

Oligonucleotide 3′-393 (SEQ ID No. 7):GGCGGCCGCGATATCGATTCATCAGTCTGAGTCAGGCCCTTC

This product was then cloned directly after PCR into the vector pCRII(Invitrogene).

A2—Construction of a cDNA Encoding the AS Form of p53

The AS form of p53 comprises a fragment encoding amino acids 1 to 366 ofthe human p53 protein supplemented with the last 19 amino acids of thealternative splicing product of the murine p53 protein.

The AS form of p53 was obtained in two stages:

PCR amplification of a fragment encoding amino acids 1 to 367 of the p53protein using the oligonucleotides 5′-1 (cf. Example A1) and 3′-367.

Oligonucleotide 3′-367 (SEQ ID No. 8):GGCGGCCGCGATATCGATTCATCAGCTCGAGTGAGC

The PCR fragment thus obtained was then cloned into the vector pCRII(Invitrogene). The fragment thus cloned has a recognition site for therestriction enzyme XhoI (fragment 1-367).

the oligonucleotides 5′-AS1, 5′-AS2, 3′-AS1 and 3′-AS2 werephosphorylated and then hybridized together in order to constitute thefragment encoding the last 19 amino acids of the alternative splicingproduct of the murine p53 protein.

5′-AS1 (SEQ ID No. 9): TCGAGCCTGCAGCCTAGAGCCTTCCAAGCCCTCATGAAGGAGG

5′-AS2 (SEQ ID No. 10): AAAGCCCAAACTGCTGATGAATCGATATCGC

3′-AS1 (SEQ ID No. 11): TGAGGGCTTGGAAGGCTCTAGGCTGCAGGC

3′-AS2 (SEQ ID No. 12): GGCCGCGATATCGATTCATCAGCAGTTTGGGCTTTCCTCCTTCA

This fragment was then inserted at the level of the XhoI site offragment 1-367 (see FIG. 2). The gene thus constructed encodes the humanvariant of the alternative splicing product of the murine p53 protein(AS).

The protein sequence thus modified is the following: SEQ ID NOS: 57-59

     364   367                                   386           393      I     I                                     I             I p53:-A H S S H L K S K K G Q S T S R H K K L M F K T E G P D S D Z AS: - A HS S L Q P R A F Q A L M K E E S P N C Z Z 367:- A H S S Z Z

A3—Construction of cDNA Encoding Various Fragments of the p53 ProteinCarrying the DNA-binding Domain

This example describes the construction of various cDNAs encodingvarious fragments of the human p53 protein, carrying all or part of theDNA binding domain of p53. These fragments are then used in theconstruction of the variants of p53. These fragments were obtained bypolymerase chain reaction on the templates described in Examples A1 andA2 by means of various oligonucleotides. The amplification reactionswere carried out under the conditions described in

Example A4.1

A3.1—Construction of a cDNA encoding the 75-325 region of p53 and itsderivative H182

This example describes the construction of a cDNA encoding amino acids75 to 325 of the wild-type human p53 protein (75-325).

This cDNA was obtained by polymerase chain reaction (PCR) on the p53 DNA(described in Example A1) with the following oligonucleotides 5′-75 and3′-325:

5′-75 (SEQ ID No. 13): GGGAAGCTTGGGCCGGGTCGACCTGCACCAGCAGCTCCT

3′-325 (SEQ ID No. 14): GGCGGCCGCGGATCCCCATCCAGTGGTTTCTT

A derivative of this fragment, carrying a point mutation on amino acid182 of the human p53 protein (cysteine→Histidine), was obtained bysite-directed mutagenesis by means of the Amersham kit, using theoligonucleotide H182 of sequence:

Oligonucleotide H182 3′ (SEQ ID No. 15): ATCTGAATGGCGCTC

This fragment was designated 75-325(H182).

A3.2—Construction of a cDNA encoding the 75-336 region of p53 and itsderivative H182

This example describes the construction of a cDNA encoding amino acids75 to 336 of the wild-type human p53 protein (75-336).

This cDNA was obtained by polymerase chain reaction (PCR) on the p53 DNA(described in Example A1) with the oligonucleotides 5′-75 (SEQ ID No.13) and 3′-336 below:

3′-336 (SEQ ID No. 16): GGCGGCCGCGGATCCTCACGCCCACGGATCTG

A derivative of this fragment, carrying a point mutation on amino acid182 of the human p53 protein (cysteine→Histidine), was obtained bysite-directed mutagenesis by means of the Amersham kit, using theoligonucleotide H182 (SEQ ID No. 15). This fragment was designated75-336(H182).

A3.3—Construction of a cDNA encoding the 75-343 region of p53

This example describes the construction of a cDNA encoding amino acids75 to 343 of the wild-type human p53 protein (75-343).

This cDNA was obtained by the polymerase chain reaction (PCR) on the DNAfor p53 (described in Example A1) with oligonucleotides 5′-75 (SEQ IDNo. 13) and 3′-343 which follows:

3-343 (SEQ ID No. 45): CGGATCCTCTCGGAACATCTCGAA

A3.4—Construction of a cDNA encoding the 75-367 region of p53 and itsderivative H182

This example describes the construction of a cDNA encoding amino acids75 to 367 of the wild-type human p53 protein (75-367).

This fragment was obtained by polymerase chain reaction (PCR) on the p53DNA described in Example A1with the oligonucleotides 5′-75 (SEQ ID No.13) and 3′-367 (SEQ ID No. 8).

The fragment thus obtained includes a recognition site for theendonuclease XhoI (75-367).

A derivative of this fragment, carrying a point mutation on amino acid182 of the human p53 protein (cysteine ->Histidine), was obtained bysite-directed mutagenesis by means of the Amersham kit, using theoligonucleotide H182 (SEQ ID No. 15). This fragment was designated75-367(H182).

A3.5—Construction of a cDNA encoding the 75-AS fragment and itsderivative H182

This example describes the construction of a cDNA encoding amino acids75 to 366 of the wild-type human p53 protein (75-366) supplemented withthe last 19 amino acids of the alternative splicing product of themurine p53 protein.

This fragment was obtained by polymerase chain reaction (PCR) on the DNAof the AS fragment described in Example A2 with the oligonucleotides5′-75 (SEQ ID No. 13) and 3′-AS2 (SEQ ID No. 12).

A derivative of this fragment, carrying a point mutation on amino acid182 of the human p53 protein (cysteine→Histidine), was obtained bysite-directed mutagenesis by means of the Amersham kit, using theoligonucleotide H182 (SEQ ID No. 15). This fragment was designated75-AS(H182).

A3.6—Construction of a cDNA encoding the 75-393 fragment of p53 and itsderivative H182

This example describes the construction of a cDNA encoding amino acids75 to 393 of the human p53 protein (75-393). This fragment was obtainedby polymerase chain reaction (PCR) on the p53 DNA described in ExampleA1 with the oligonucleotides 5′-75 (SEQ ID No. 13) and 3′-393 (SEQ IDNo. 7).

A derivative of this fragment, carrying a point mutation on amino acid182 of the human p53 protein (cysteine→Histidine), was obtained bysite-directed mutagenesis by means of the Amersham kit, using theoligonucleotide H182 (SEQ ID No. 15). This fragment was designated75-393(H182).

A4—Construction of cDNA Encoding Various Fragments Carrying aTranscription-activating Domain (Transactivating Domain)

This example describes the construction of various cDNA encoding variousfragments carrying a transactivating domain. These fragments are thenused in the construction of the variants of p53.

A4.1—PCR reactions

The various fragments were obtained by polymerase chain reaction onvarious templates by means of various oligonucleotides. Theamplification reactions were carried out under the following conditions:Amplitaq DNA polymerase enzyme (Perkin-Elmer) in the buffer provided bythe supplier, with a dNTP concentration of 0.2 mM, 100 ng of templateand 500 ng of each of the two oligonucleotides.

cycle: 2 min at 91° C.

cycles:

1 min at 91° C.

min at 55° C.

min at 72° C.

cycle: 5 min at 72° C.

A4.2—Construction of a cDNA encoding the 411-490 region of the viralprotein VP16 (VP16 TA)

This example describes the construction of a cDNA coding amino acids411-490 of the viral protein VP16 (VP16 TA). This region carries thetransactivating domain of this protein.

The transactivating fragment derived from the 10 viral protein VP16(411-490) of the herpes simplex virus was obtained by polymerase chainreaction (PCR) using the conditions defined above (cf. A4.1) and thefollowing oligonucleotides 5′-VP16 and 3′-VP16:

5′-VP16 (SEQ ID No. 17): AAGCTTGAATTCGTTAACATGTCCACGGCCCCCCCGACC

3′-VP16 (SEQ ID No. 18): GGTCGACCACCGTACTCGTCAAT and 100 ng of theplasmid pUHD15-1 (Gossen & Bujard, Proc. Natl. Acad. Sci. USA 89 (1992)5547)

The fragment thus obtained comprises 334 base pairs whose sequence isrepresented SEQ ID No. 2. It comprises, in its N-terminal part, amethionine residue provided by the site for initiation of transcription(ATG), added during the PCR cloning step.

A4.2—Construction of cDNA encoding various fragments capable ofrecruiting the transcription activating domain (transactivating domain)of an endogenous p53 protein.

A4.2.1—Construction of a cDNA encoding a single-chain antibody capableof binding the p53 protein (ScFv 421)

This example describes the construction of a cDNA encoding asingle-chain antibody capable of binding the p53 protein (ScFv 421).This construct, which is expressed at the intracellular level, should becapable of binding a wild-type or mutant endogenous p53 protein in orderto recruit its transactivating domain.

The cDNA encoding ScFv 421 (Patent Application PCT/FR96/00477) can beextracted in the form of an NcoI/NotI fragment which comprises a sitefor initiation of translation (ATG) and no sequence for termination oftranslation.

The fragment thus obtained comprises 766 base pairs whose sequence isgiven SEQ ID No. 46.

A4.2.2—Construction of a cDNA encoding the 325-360 region of thewild-type p53 protein (325-360)

This example describes the construction of a cDNA encoding amino acids325-360 of the wild-type p53 protein (325-360). This region carries theoligomerization domain of this protein. This construct, which isexpressed at the intracellular level, should be capable of binding awild-type or mutant endogenous p53 protein in order to recruit itstransactivating domain.

This oligomerization domain, which is derived from the human wild-typep53 protein (325-360), was obtained by polymerase chain reaction (PCR)using the conditions defined above (Cf A4.1) and the ligonucleotides5′-325 and 3′-360 which follow:

5′-325 (SEQ ID No. 47): AAGCTTGAATTCGTTAACGCCACCATGGGAGAATATTTCACCCTT

3′-360 (SEQ ID No. 48): GGGTCGACCTGGCTCCTTCCCAGC

on 100 ng of p53 DNA (described in Example A1).

The fragment thus obtained comprises 141 base pairs whose sequence isgiven SEQ ID No. 49. It comprises, in its N-terminal part, a methionineresidue provided by the site for initiation of translation (ATG), addedduring the PCR cloning step and no sequence for termination oftranslation.

A4.2.3—Construction of a cDNA encoding the 325-393 region of thewild-type p53 protein (325-393)

This example describes the construction of a cDNA encoding amino acids325-393 of the wild-type p53 protein (325-393). This region carries theoligomerization domain of this protein. This construct, which isexpressed at the intracellular level, should be capable of binding awild-type or mutant endogenous p53 protein in order to recruit itstransactivating domain.

This oligomerization domain, which is derived from the human wild-typep53 protein (325-360), was obtained by polymerase chain reaction (PCR)using the conditions defined above (Cf A4.1) and the oligonucleotides5′-325 (SEQ ID No. 47) and 3′-393.2 which follows:

3′-393.2 (SEQ ID No. 50): GGGTCGACCGTCTGAGTCAGGCCCTTC

on 100 ng of the p53 DNA (described in Example A1).

The fragment thus obtained comprises 243 base pairs whose sequence isgiven SEQ ID No. 51. It comprises, in its N-terminal part, a methionineresidue provided by the site for inititation of translation (ATG), addedduring the PCR cloning step and no sequence for termination oftranslation.

A5—Construction of cDNA Encoding Various Fragments Carrying anOligomerization Domain

This example describes the construction of various cDNAs encodingvarious fragments carrying an oligomerization domain. These fragmentsare then used in the construction of the p53 variants. These fragmentswere obtained by polymerase chain reaction on various templates (p53 forthe homologous domain and templates of various origins for theheterologous, especially artificial, oligomerization domains) by meansof various oligonucleotides. The amplification reactions were carriedout under the conditions described in A4.1 above.

A5.1—Construction of a cDNA comprising an artificial oligomerizationdomain.

This example describes the construction of a cDNA comprising anartificial oligomerization domain, consisting of an artificial leucinezipper. This cDNA was then used for the construction of variants fromfragments 75-325 (Example A3.1) and 75-336 (Example A3.2) of the humanp53 protein and of their derivatives modified at the level of thecysteine 182.

This cDNA was constructed from the following 6 oligonucleotides:

lz1-5′ (SEQ ID No. 19): GATCTGAAGGCCCTCAAGGAGAAGCTGAAGGCC

lz2-5′ (SEQ ID No. 20): CTGGAGGAGAAGCTGAAGGCCCTGGAGGAGAAGCTG

lz3-5′ (SEQ ID No. 21) AAGGCACTAGTGGGGGAGCGATGATGAATCGATATCGC

lz1-3′ (SEQ ID No. 22): CTCCTCCAGGGCCTTCAGCTTCTCCTTGAGGGCCTTCA

lz2-3′ (SEQ ID No. 23): TAGTGCCTTCAGCTTCTCCTCCAGGGCCTTCAGCTT

lz3-3′ (SEQ ID No. 24): GGCCGCGATATCGATTCATCATCGCTCCCCCAC

These oligonucleotides were synthesized by means of an automatic DNAsynthesizer, using the chemistry of phosphoramidites. These sixoligonucleotides exhibit complementarity in pairs (lz1-5′/lz-1-3′,lz2-5′/lz2-3′, lz3-5′/lz3-3′) and overlapping complementarities(lz1-3′/lz2-5′, lz2-3′/lz3-5′) which allow the oligomerization domain tobe obtained simply by hybridization and ligation. The resulting LZsequence is represented SEQ ID No. 1.

A5.2—Construction of a cDNA comprising the natural oligomerizationdomain of human p53.

This example describes the construction of a cDNA comprising the naturaloligomerization domain of human p53. This cDNA is represented by thefragment encoding amino acids 325 to 356 of p53 which is contained inthe constructs 75-367 (Example A3.3), 75-AS (Example A3.4), 75-393(Example A3.5) and of their derivatives modified at the level of thecysteine 182.

Example B

Construction of the Genes Encoding Various Variants of the p53 Protein

B1—Cloning of the Various Fragments of p53

Each of the various fragments obtained by PCR described in Example A wascloned after PCR into the vector pBC SK+ (Stratagene) using therecognition sites for the restriction enzymes HindIII and NotI (FIG. 3).

The products of these constructions carry the following numbers:

75-325 → pEC 104 75-336 → pEC 106 75-343 → pEC 171 75-367 → pEC 13175-AS → pEC 132 75-393 → pEC 133

From these products and by site-directed mutagenesis using theoligonucleotide-directed in vitro mutagenesis system (Amersham) and theoligonucleotide H182, the corresponding constructs carrying a histidinein position 182 were obtained. These constructs carry the followingnumbers:

75-325(H182) → pEC 134 75-336(H182) → PEC 135 75-367(H182) → pEC 13675-AS(H182) → pEC 137 75-393(H182) → pEC 138

B2—Fusion of the Leucine Aipper to Fragments 75-325, 75-336 and 75-343and to their Variant H182

The oligonucleotides constituting the leucine zipper (lz1-5′, lz1-3′,lz2-5′, lz2-3′, lz3-5′and lz3-3′) were phosphorylated with the aid of T4kinase and they were all hybridized together and inserted into thevectors pEC 104, 106, 134, 135 and 171 previously digested with therestriction enzymes BamHI and NotI (FIG. 4).

The products of these constructions carry the following numbers:

75-325-lz → pEC 107 75-336-lz → pEC 110 75-343-lz → pEC 17475-325(H182)-lz → pEC 139 75-336(H182)-lz → pEC 140

B3—Fusion of the Transcription-activating Domain to the Entire p53Fragments

The final products were obtained by a three-partner ligation carried outin the following manner (FIG. 5):

The transcription-activating domain derived from VP16 which is describedin Example A3 was prepared by enzymatic digestion of the PCR productswith the restriction enzymes HindIII and SalI.

The various p53 fragments obtained above (75-325-lz, 75-336-lz,75-343-lz, 75-AS, 75-367, 75-393 and their H182 variants) were isolatedafter enzymatic digestion of the plasmids containing them with therestriction enzymes SalI and NotI.

The possible combinations (activating domain/p53) were constituted andinserted simultaneously into the vector pBC SK+(Stratagene) previouslydigested with the restriction enzymes HindIII and NotI.

The products of these constructions carry the following numbers:

VP16-75-325-1z V-325 → PEC 114 (SEQ ID NOS: 25 and 26) VP16-75-336-1zV-336 → pEC 116 (SEQ ID NOS: 27 and 28) VP16-75-367 V-367 → pEC 141 (SEQID NOS: 29 and 30) VP16-75-AS V-AS → pEC 143 (SEQ ID NOS: 31 and 32)VP16-75-393 V-393 → pEC 145 (SEQ ID NOS: 33 and 34) VP16-75-343 V-343 →pEC 175 (SEQ ID NOS: 35 and 36) VP16-75-325(H182)-1z V-325H → pEC 147VP16-75-336(H182)-1z V-336H → pEC 149 VP16-75-367(H182) V-367H → pEC 151VP16-75-AS(H182) V-ASH → pEC 153 VP16-75-393(H182) V-393H → pEC 155

The corresponding products, carrying a domain which specifically binds atransactivator or a transactivating complex in place of the VP16 domain,are constructed in the same manner. These constructs are designatedbelow:

ScFv-75-325-1z S-325 → 176 (SEQ ID NOS: 37 and 38) ScFv-75-336-1z S-336ScFv-75-367 S-367 ScFv-75-AS S-AS ScFv-75-393 S-393 ScFv-75-325(H182)-1zS-325H ScFv-75-336(H152)-1z S-336H ScFv-75-367(H182) S-367HScFv-75-AS(H182) S-ASH ScFv-75-393(H182) S-393H (325-393)-75-325-1z393-325 → pEC 177 (SEQ ID NOS: 39 and 40) (325-393)-75-367 393-367(325-393)-75-AS 393-AS (325-393)-75-393 393-393(325-393)-75-325(H182)-1z 393-325H (325-393)-75-336(H182)-1z 393-336H(325-393)-75-367(H182) 393-367H (325-393)-75-As(H182) 393-ASH(325-393)-75-393(H182) 393-393H (325-360)-75-325-1z 360-325 → pEC 178(SEQ ID NOS: 41 and 42) (325-360)-75-336-1z 360-336 (325-360)-75-367360-367 (325-360)-75-AS 360-AS (325-360)-75-393 360-393(325-360)-75-325(H182)-1z 360-325H (325-360)-75-336(H182)-1z 360-336H(325-360)-75-367 (H182) 360-367H (325-360)-75-AS(H182) 360-ASH(325-360)-75-393(H182) 360-393H

The products containing the 325-360 domain of p53, as a replacement forthe transactivating domain (1-74) may be subject to the addition of asynthetic separator (Hinge) obtained by insertion, at the SalI site, ofa DNA fragment obtained by hybridization of the pair of complementarysynthetic oligonucleotides Hinge-up and Hinge-down which follow:

Hinge-up (SEQ ID No. 52):TCGAGGAGGTGGTGGCTCTGGAGGCGGAGGATCCGGCGGTGGAGGTTC

Hinge-down (SEQ ID No. 53):TCGAGAACCCCTACCGCCGGATCCTCCGCCTCCAGAGCCACCACCTCC

The resulting Hinge double-stranded DNA sequence is the followingTCGAGGAGGTGGTGGCTCTGGAGGCGGAGGATCCGGCGGTGGAGGTTC SEQ ID NO:52CCTCCACCACCGAGACCTCCGCCTCCTAGGCCGCCATCCCCAAGAGCT and the correspondingprotein sequence is (SEQ ID No. 54):Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser

The corresponding products are designated below:

(325-360)-Hinge-75-325-1z 360h-325 → pEC 179 (SEQ ID NOS: 43 and 44)(325-360)-Hinge-75-336-1z 360h-336 (325-360)-Hinge-75-367 360h-367(325-360)-Hinge-75-AS 360h-AS (325-360)-Hinge-75-393 360h-393(325-360)-Hinge-75-325(H182)-1z 360h-325h(325-360)-Hinge-75-336(H182)-1z 360h-336H (325-360)-Hinge-75-367(H182)360h-367h (325-360)-Hinge-75-AS(H182) 360h-ASH(325-360)-Hinge-75-393(H182) 360h-393H

Example C

Constructiozi of Expression Vectors for the Variants of p53

This example describes the construction of vectors which can be used forthe transfer of the nucleic acids of the invention in vitro or in vivo.

C1—Construction of Plasmid Vectors

For the construction of plasmid vectors, 2 types of vectors were used.

The vector pSV2, described in DNA Cloning, A practical approach Vol.2,D. M. Glover (Ed) IRL Press, Oxford, Washington D.C., 1985. This vectoris a eukaryotic expression vector. The nucleic acids encoding thevariants were inserted into this vector in the form of HpaI-EcoRVfragments. They are thus placed under the control of the promoter andthe enhancer of the SV40 virus.

The vector pcDNA3 (Invitrogen). It is also a eukaryotic expressionvector. The nucleic acids encoding the variants of the invention arethus placed, in this vector, under the control of the early CMVpromoter. All the constructs described in Example B3 were introducedinto this vector in the form of a HindIII/NotI fragment so as to betested in the various systems for evaluation in vivo.

C2—Construction of Viral Vectors

According to a specific mode, the invention consists in the constructionand the use of viral vectors which allow the transfer and the expressionin vivo of the nucleic acids as defined above.

As regards more particularly adenoviruses, various serotypes, whosestructure and properties vary somewhat, have been characterized. Amongthese serotypes, the use of the type 2 or 5 human adenoviruses (Ad 2 orAd 5) or of the adenoviruses of animal origin (see applicationWO94/26914) is preferred within the framework of the present invention.Among the adenoviruses of animal origin which can be used within theframework of the present invention, there may be mentioned adenovirusesof canine, bovine, murine (example: MAV1, Beard et al., Virology 75(1990) 81), ovine, porcine, avian or alternatively simian (example: SAV)origin. Preferably, the adenovirus of animal origin is a canineadenovirus, more preferably a CAV2 adenovirus [Manhattan strain orA26/61 (ATCC VR-800) for example]. Preferably, adenoviruses of human orcanine or mixed origin are used within the framework of the invention.

Preferably, the defective adenoviruses of the invention comprise theITRs, a sequence allowing the encapsidation and a nucleic acid accordingto the invention. Still more preferably, in the genome of theadenoviruses of the invention, at least the E1 region is nonfunctional.The viral gene considered can be rendered non-functional by anytechnique known to persons skilled in the art, and especially by totalsuppression, by substitution or partial deletion, or by addition of oneor more bases in the gene(s) considered. Such modifications can beobtained in vitro (on the isolated DNA) or in situ, for example by meansof genetic engineering techniques, or alternatively by treating withmutagenic agents. Other regions can also be modified, and especially theregion E3 (WO95/02697), E2 (WO94/28938), E4 (WO94/28152, WO94/12649,WO95/02697) and L5 (WO95/02697). According to a preferred embodiment,the adenovirus according to the invention comprises a deletion in the E1and E4 regions. According to another preferred embodiment, it comprisesa deletion in the E1 region, at the level of which are inserted the E4region and the nucleic acid of the invention (cf. FR94/13355). In theviruses of the invention, the deletion in the E1 region preferablyextends from nucleotides 455 to 3329 on the sequence of the Ad5adenovirus.

The defective recombinant adenoviruses according to the invention can beprepared by any technique known to persons skilled in the art (Levreroet al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917).In particular, they can be prepared by homologous recombination betweenan adenovirus and a plasmid carrying, inter alia, the DNA sequence ofinterest. The homologous recombination occurs after co-transfection ofthe said adenoviruses and plasmid into an appropriate cell line. Thecell line used should preferably (i) be transformable by the saidelements, and (ii) contain the sequences capable of complementing thedefective adenovirus genome part, preferably in integrated form in orderto avoid risks of recombination. As an example of a cell line, there maybe mentioned the human embryonic kidney line 293 (Graham et al., J. Gen.Virol. 36 (1977) 59) which contains especially, integrated in itsgenome, the left hand part of the genome of an Ad5 adenovirus (12%) orlines capable of complementing the E1 and E4 functions as describedespecially in applications Nos. WO 94/26914 and WO95/02697.

Next, the adenoviruses which have multiplied are recovered and purifiedaccording to conventional molecular biology techniques as illustrated inthe examples.

As regards the adeno-associated viruses (AAV), they are relatively smallDNA viruses which become integrated into the genome of the cells whichthey infect, in a stable and site-specific manner. They are capable ofinfecting a broad spectrum of cells, without inducing any effect on cellgrowth, morphology or differentiation. Moreover, they do not seem to beinvolved in pathologies in man. The genome of the AAVs has been cloned,sequenced and characterized. It comprises about 4700 bases and contains,at each end, an inverted repeat region (ITR) of about 145 bases whichserves as replication origin for the virus. The remainder of the genomeis divided into 2 essential regions carrying the encapsidationfunctions: the left-hand part of the genome, which contains the rep geneinvolved in the viral replication and the expression of the viral genes;the right-hand part of the genome, which contains the cap gene encodingthe virus capsid proteins.

The use of vectors derived from AAVs for the transfer of genes in vitroand in vivo has been described in the literature (see especially WO91/18088; WO 93/09239; U.S. Pat. Nos. 4,797,368, 5,139,941, EP 488 528).These applications describe various constructs derived from AAVs, fromwhich the rep and/or cap genes are deleted and replaced by a gene ofinterest, and their use for the transfer in vitro (on cells in culture)or in vivo (directly in an organism) of the said gene of interest. Thedefective recombinant AAVs according to the invention can be prepared byco-transfection, into a cell line infected by a human helper virus (forexample an adenovirus), of a plasmid containing a nucleic sequence ofthe invention of interest bordered by two AAV inverted repeat regions(ITR), and of a plasmid carrying the AAV encapsidation genes (rep andcap genes). An example of a cell line which can be used is line 293. Therecombinant AAVs produced are then purified by conventional techniques.

As regards the herpesviruses and the retroviruses, the construction ofrecombinant vectors has been widely described in the literature: seeespecially Breakfield et al., New Biologist 3 (1991) 203; EP 453242, EP178220, Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick,BioTechnology 3 (1985) 689, and the like. In particular, theretroviruses are integrative viruses which selectively infect thedividing cells. They therefore constitute vectors of interest for cancerapplications. The genome of retroviruses essentially comprises two LTRs,an encapsidation sequence and three coding regions (gag, pol and env).In the recombinant vectors derived from retroviruses, the gag, pol andenv genes are generally deleted, completely or partly, and replaced by aheterologous nucleic acid sequence of interest. These vectors can beprepared from various types of retroviruses such as especially MoMuLV(murine Moloney laukaemia virus, also called MoMLV), MSV (murine Moloneysarcoma virus), HaSV (Harvey sarcoma virus), SNV (spleen necrosisvirus), RSV (Rous sarcoma virus) or alternatively Friend's virus.

To construct the recombinant retroviruses according to the inventioncontaining a nucleic acid according to the invention, a plasmidcontaining especially the LTRs, the encapsidation sequence and the saidnucleic acid is constructed and then used to transfect a so-calledencapsidation cell line capable of providing in trans the retroviralfunctions which are deficient in the plasmid. Generally, theencapsidation lines are therefore capable of expressing the gag, pol andenv genes. Such encapsidation lines have been described in the priorart, and especially the PA317 line (U.S. Pat. No. 4,861,719), thePsiCRIP line (WO 90/02806) and the GP+envAm-12 line (WO 89/07150).Moreover, the recombinant retroviruses may contain modifications in theLTRs so as to suppress the transcriptional activity, as well as extendedencapsidation sequences containing a portion of the gag gene (Bender etal., J. Virol. 61 (1987) 1639). The recombinant retroviruses producedare then purified by conventional techniques.

For the implementation of the present invention, it is most particularlyadvantageous to use a defective recombinant retrovirus or adenovirus.These vectors possess, indeed, properties which are particularlyadvantageous for the transfer of genes into tumour cells.

C3—Chemical Vectors

Among the synthetic vectors developed, it is preferred to use within theframework of the invention cationic polymers of the polylysine type(LKLK)n, (LKKL)n, polyethyleneimine type and DEAE dextran type oralternatively cationic lipids or lipofectants. They possess the propertyof condensing DNA and of promoting its association with the cellmembrane. Among the latter, there may be mentioned lipopolyamines(lipofectamine, transfectam and the like), various cationic or neutrallipids (DOTMA, DOGS, DOPE and the like) as well as peptides of nuclearorigin. In addition, the concept of targeted transfection was developed,mediated by a receptor, which takes advantage of the principle ofcondensing DNA by means of the cationic polymer while directing theattachment of the complex to the membrane by virtue of a chemical linkbetween the cationic polymer and the ligand for a membrane receptor,present at the surface of the cell type which it is desired to graft.The targeting of the receptor for transferrin and for insulin or of thereceptor for the asialoglycoproteins of the hepatocytes has thus beendescribed. The preparation of a composition according to the inventionusing such a chemical vector is performed according to any techniqueknown to persons skilled in the art, generally by simply bringing thevarious components into contact.

Example D

Functional Evaluation of the Variants of p53

The variants of p53 according to the invention were evaluated in acellular test for the following criteria:

binding to a specific double-stranded DNA sequence

transactivating function

antiproliferative activity

apoptotic activity

oncogenic potential associated with some p53 mutations

The constructs used more particularly for this evaluation are theconstructs V-325, V-336, V-343 and AS described in Example B.

D1 Recognition of specific double-stranded DNA sequences by the hybridmolecules of the invention

D1.1 Production of the hybrid molecules

The cDNA of the wild-type p53 was cloned into the vector pBlueBacIII(Invitrogen) at the BamHI site. By inserting into the plasmid pAcHLT-A(Pharmingen) the fragment containing the V325 cDNA obtained by digestingthe plasmid pEC114 with the enzymes EcoRI and NotI, a vector wasgenerated which allows the production of a recombinant baculovirus whoseaim is the expression of a V325 protein tagged at the N-terminus with apeptide sequence containing, inter alia, a chain of 6 histidineresidues. Using these vectors, recombinant baculoviruses were producedand purified according to the manufacturer's instructions (Invitrogen,Pharmingen). The two proteins were purified to homogeneity using nuclearextracts of SF9 insect cells infected with their respectivebaculoviruses, the nuclear extracts being obtained according to theprocedure described by Delphin et al. (C. Delphin, Eur. J. Biochem.,223, 683-692, 1994).

The wild-type p53 is purified by immuno-affinity on the monoclonalantibody pAb421 (Oncogene Sciences, Ab-1) according to the followingprotocol: the nuclear extract of the infected cells is incubated for 3 hat 4° C. with a protein A-agarose gel onto which the antibody pAb421 hasbeen covalently coupled. After extensive washing of the gel with a 50 mMTris-HCl buffer pH 7.8, containing 1 M KCl and protease inhibitors, thep53 protein is eluted by the peptide corresponding to the epitoperecognized by this antibody on p53 (KKGQSTSRHK), this peptide being usedat a concentration of 5 mg/ml in the solution used for the washing.After concentration on Centricon-30 (Amicon Grace), the eluted p53 isseparated from the peptide and purified to homogeneity by gel permeationon a Superose 6 HR10/30 column equilibrated with 50 mM Tris-HCl, pH 7.5,0.2 M NaCl, 0.1 mM EGTA, 0.1 mM ZnCl₂, 10 mM DTT, 0.1 mM PMSF, 0.1%NP-40, 5% glycerol. The fractions containing p53 are aliquoted andimmediately frozen at −80° C. until they are used.

The V325 protein, tagged at the N-terminals with a peptide sequencecontaining, inter alia, a chain of 6 histidine residues, henceforthcalled HisV325, was purified by a procedure adapted from Hochuli et al.(Bio/Technology Vol. 6 (1988) 1321). Before being applied to theNickel-NTA agarose gel, the nuclear extract of the infected cells isdesalted on a PD10 column (Pharmacia) equilibrated in 50 mM sodiumphosphate buffer pH 8, containing 5 mM β-mercapto-ethanol, 0.1% NP-40and a cocktail of protease inhibitors. The incubation of the nuclearextract with the nickel NTA agarose gel was carried out in this bufferfor 1 h at 4° C., with stirring. The gel is then washed extensively withthe same buffer at pH 6. The HisV325 protein is eluted with 0.3 Mimidazole in the latter buffer after washing the gel in 0.1 M imidazole.The fractions containing HisV325 are aliquoted and immediately frozen at−80° C. until they are used.

D1.2 Construction of the specific doubled-stranded DNA sequence

The specific double-stranded DNA sequence used in this experimentconsists of two synthetic oligonucleotides whose sequence is thefollowing:

Oligo 5568 (SEQ ID No. 55): GATCCGAACATGTCCCAACATGTTGA

Oligo 5569 (SEQ ID No. 56): AGCTTCAACATGTTGGGACATGGTTCG

These two synthetic oligonucleotides were labelled with phosphorus-33 bya 30-min incubation at 37° C. of 5 pmol of each oligonucleotide in 20 μlof the following reaction medium:

Tris-HCl pH 7.6  50 mM MgCl₂   10 mM dithiothreitol  5 mM Spermidine 100μM EDTA 100 μM [γ-³³P]ATP (Amersham)  50 μ Ci (1000-3000 Ci/mmol) T4kinase (Boehringer)  10 U

Next, the two oligonucleotides thus labelled were hybridized in thepresence of 100 mM NaCl in order to reconstitute the followingdouble-stranded sequence WAF-RE containing the specific sequencerecognized by p53 in the promoter region of the WAF-1 gene (W. S.El-Deiry, Cell Vol75 (1993) 817):

GATCCGAACATGTCCCAACATGTTGA GCTTGTACAGGGTTGTACAACTTCGA

D1.3 Recognition of the double-stranded sequence WAF-RE by the hybridmolecules of the invention.

To demonstrate a specific recognition of the double-stranded sequenceWAF-RE by the hybrid molecules of the invention, gel retardationexperiments were carried out on the principle described below. The DNAbinding reaction is carried out in 25 μl of reaction medium (20 mMTris-HCl pH 7.5, 5 mM MgCl₂, 0.05 mM ZnCl₂, 5 mM dithiotreitol, 0.1mg/ml BSA, 10% glycerol, 1% Nonidet P-40, 0.1 M NaCl, 2 μg/ml aprotinin,2 μg/ml E-64, 2 μg/ml leupeptin, 2 μg/ml pepstatin) by adding thesequence WAF-RE (2.4×10⁻⁹ M) prepared according to the precedingexample, 1.2×10⁻⁶ M of the cold competitor oligonucleotide (Promega)used to eliminate nonspecific binding and 30 ng of hybrid molecules tobe tested in the presence or otherwise of wild-type p53 (between 3 and30 ng), it being possible for the wild-type p53 to be activated, for itsspecific DNA binding activity, by 300 ng of pAb421 antibody (T. R. Hupp,Cell Vol 71 (1992) 875). The reaction mixtures are incubated for 30minutes on ice and the final mixtures are subjected to a 4% nativepolyacrylamide gel electrophoresis with migration at 200 V and 16° C.The gel is then dried and autoradiographed.

The result of a representative experiment of competition betweenwild-type p53 and HisV325 in gel retardation is presented in FIG. 6.This result shows that His-V325 recognizes the double-stranded sequenceWAF-RE with a comparable affinity to that of the wild-type p53. It willbe noted that HisV325 gives a predominant band in gel retardation whichmigrates faster than that obtained with the wild-type p53. This couldindicate that HisV325 binds in the form of a diner. Furthermore, asexpected, this band is neither displaced nor amplified by the presenceof pAb421. Finally, in the absence of pAb421, the wild-type p53 bindsmuch less RE-WAF than does V325.

D2—Evaluation of the Transactivating Function

The transactivating function of the constructs was evaluated in atransactivating system in vivo in SAOS-2 (human osteosarcoma) cellsdeficient for the two alleles for the p53 protein (cells accessible atATCC under the number HTB85) and in the tumour lines H358 (Maxwell &Roth, Oncogene 8 (1993), 3421) and HeLa (ATCC CCL 2). This system isbased on the use of a reporter gene which can be assayed enzymaticallyand which is placed in dependence on a promoter containing thenucleotide units for specific recognition by the wild-type form of p53(cf. experimental procedures).

In this test, the reporter gene is the CAT (chloramphenicol-acetyltransferase) gene and the sequence for recognition by p53 is theconsensus sequence (p53RE) defined by Funk et al. (Mol. Cell. Biol. 12(1992) 2866).

The evaluation of this function was carried out in comparison with thatof the wild-type protein for three different types of criteria.

D2.1—Transactivating activity in response dose

The cells (3.5×10⁵) are inoculated in Petri dishes 6 cm in diametercontaining 3 ml of DMEM medium (Gibco BRL) supplemented with 10%heat-inactivated foetal calf serum, and they are cultured overnight in aCO₂ (5%) incubator at 37° C. The various constructs are then transfectedusing lipofectAMINE (Gibco BRL) as transfection agent in the followingmanner: 3 μg of total plasmid are incubated (of which 0.5 μg of thereporter plasmid) and 10 μl of lipofectAMINE for 30 min with 3 ml ofOpti-MEM medium (Gibco BRL) without serum (transfection mixture). Duringthis period, the cells are rinsed twice with PBS and then incubated for4 hours at 37° C. with the transfection mixture, after which the latteris aspirated and replaced with 3 ml of DMEM medium supplemented with 10%heat-inactivated foetal calf serum and the cells allowed to grow againfor 48 hours at 37° C.

Procedure for Assaying the CAT Activity

48 hours after transfection, the cells are washed once with PBS and thenscraped and recovered in 100 μl of 0.25 M Tris buffer pH 8 and lysed bythree freeze-thaw cycles in an ethanol/solid carbon dioxide bath. Thetotal cellular extract thus obtained is centrifuged for 15 min at 10,000rpm and the supernatant recovered for the assay of activity. The latteris carried out by adding 20 μl of cellular extract to 130 μl of areaction mixture whose final composition is the following:

Acetyl-coenzyme A 0.4 mM Chloramphenicol,  23 μM (200 nCi)D-threo-(dichloroacetyl-1,2-¹⁴C) Tris 0.18M pH 8

After incubating for 1 hour at 37° C., the reaction products areextracted with 250 μl of ethyl acetate, of which 20 μl are placed on asilica plate (thin-layer chromatography) which is allowed to run in amixture containing 95% chloroform and 5% methanol. The chromatographyplate thus obtained is finally developed using an instant imager(Packard instruments) which makes it possible to calculate the ratio ofthe various products of acetylation, which ratio reflects the activityof the enzyme chloramphenicol acetyl-transferase and therefore thetransactivating activity of the various constructs.

The results obtained in the SAOS-2 line with the constructs placed underthe control of the CMV promoter (pcDNA3) and presented in FIGS. 7 and 8show the following properties for each of the constructs:

the p53 protein has a dose-dependent activity which tends towardssaturation for high doses (from 100 ng of plasmid). This saturation mayreflect the need for cofactors which could be limiting under theseconditions.

the AS protein conserves the transcription-activating capacity of thewild-type protein.

the V-325, V-336 and V-343 proteins have, just like the p53 protein, atransactivating activity which does not appear, for its part, saturableat high doses. It is therefore possible that this apparent lack ofsaturation may lead to an overall increase in activity. It appears, inaddition, that the construct V-325 is more active than its homologuesV-336 and V-343, which suggests that the chimeric proteins carrying the75-325 region are particularly advantageous.

With the aim of confirming these properties, a similar experiment wascarried out in the tumour line H 358 which, just like the SAOS-2 line,is deficient for the two alleles of the p53 gene. In this experiment,each transfection was carried out with 50 ng of each of the constructsplaced in dependence on the CMV promoter. The results presented in Table1 show clearly that the two variants V-325 and V-336 show an improvedtransactivating activity compared with that of the wild-type p53protein, with again a better activity for the variant V-325.

TABLE 1 Transactivating activity in the cells of the tumour line H 358.pCDNA 3 wild-type p53 V-325 V-336 Relative CAT 1 6 25 16 activity

These two tests confirm that the variants of the invention have at leastone of the properties of the improved p53.

In order to verify that this difference in activity is not due to adifference in expression, but indeed to an increased activity of thevariants of the invention, the level of expression of the wild-type p53protein and of the variants V-325, V-336 and V-343 was analysed inSAOS-2 cells. To do this, the cells are transfected with 3 μg of each ofthe plasmids used in the preceding experiment, and recovered 24 hoursand 48 hours after transfection. After two washes in PBS buffer (GibcoBRL), the cells are lysed for 15 minutes at 4° C. in 50 μl of RIPAbuffer (10 mM Tris-Hcl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Nonidet-P40,1% sodium deoxycholate, 0.1% sodium dodecyl sulphate) supplemented with2 mM PMSF, 20 μg/ml aprotinin, 2 μg/ml E64, 2 μg/ml leupeptin and 2μg/ml pepstatin. After 15 min of centrifugation at 15,000 rpm, thesupernatants are collected, supplemented with migration buffer (LaemmliU. K., Nature, 227, 680-685, 1970) and subjected to electrophoresis on a10% polyacrylamide gel in denaturing medium at 200 V according to theprotocol previously described (Laemmli U. K., Nature, 227, 680-685,1970). Next, the proteins are transferred onto PVDF membrane (NENResearch Products) using the NOVEX semidry transfer system according tothe manufacturer's recommendations, and revealed with the aid of themonoclonal antibody pAb 240 (Oncogene Sciences, Ab-3) and of a secondaryantibody (anti-mouse rabbit antibody) coupled to peroxidase (NordicImmunology) using the ECL kit (Amersham).

The result of this experiment which is presented in FIG. 9 shows thatthe variants V-325 and V-336 are expressed at a comparable level to thatof the wild-type p53 protein and that the variant V-343 appears to beslightly better expressed than the previous ones. Furthermore,comparison of the expression levels at 24 and 48 hours appears toindicate that the relative stability of each of the constructs issimilar. This result therefore shows that the increased activity of thevariants of the invention V-325 and V-336 is not due to a betterexpression but probably to an increased transcriptional activatingpotential, contrary to the V-343 variant.

Subsequently, and with the aim of confirming this capacity of thevariants of the invention to activate a gene placed under the control ofa wild-type p53 recognition element, the study of the activation ofendogenous genes was carried out by looking at the expression of thehdm2 and WAF1 genes, which are normally induced by the p53 protein.

This experiment was carried out in the EB cell line (colon cancer)deficient for the two alleles encoding the p53 protein (Shaw et al.,PNAS 89 (1992) 4495). A stable clone expressing the p53 protein underthe control of the inducible metallothionein promoter was constructedfrom this line (clone EB-1 (Shaw et al., PNAS 89 (1992) 4495)).Likewise, another stable clone expressing the V-325 protein under thecontrol of the inducible metallothionein promoter was constructed usinga plasmid derived from the vector pmIMTli (obtained from P. Shaw) byinsertion of the cDNA encoding the V-325 protein at the EcoRI-NotI sites(pmIMTli-V325). To do this, the EB cells (3.5×10⁵ cells) weretransfected with 1.3 μg of the plasmid pmIMTli-V325 and 200 ng ofplasmid pcDNA3 following the procedure described above and the stableclones were selected after transfection by growing in a mediumcontaining 800 μg/ml of geneticin. A clone expressing V-325 in aninducible manner at a level comparable to the expression of the p53protein in the EB-1 clone was selected (EB-V325 clone).

The EB-1 and EB-V325 clones as well as the EB parental cells (10⁶ cells)were subjected to treatment with ZnCl₂ (200 μM) and cell extracts wereprepared at various times and subjected to electrophoresis and membranetransfer as described above. The transferred proteins were revealed bythree different antibodies; the monoclonal antibody pAb240 directedagainst the p53 protein, and two polyclonal antibodies, one directedagainst the hdm2 protein and the other against the WAFI protein. Theresults of this experiment, presented in FIG. 10, show that: 1) the p53protein, absent from the EB, EB-V325 and EB-1 cells in the absence ofinduction, is expressed in the EB-1 clone as early as 4 hours after thestart of the zinc treatment, and the variant V-325 is expressed in thesame manner in the EB-V325 clone, 2) the WAF1 protein whose expressionappears to be induced in the EB cells by the zinc treatment 4 hoursafter starting it, sees its expression prolonged up to 16 hours in theEB-1 and EB-V325 clones, and 3) the induction of the hdm2 protein isobservable only in the EB-1 and EB-V325 clones, with an increasedexpression in the EB-V325 clone.

These results show that the activation of transcription by the V-325variant results in the induction of the expression of genes normallyinduced by the wild-type p53 protein, and that this variant indeedexhibits an increased activity in a physiological context relative tothe wild-type p53 protein.

D2.2—Effect of the E6 protein (HPV18) on the transactivating function

The procedures used are identical to those described in Example D1.1. Inthis transfection experiment, the constructs placed under the control ofthe CMV promoter (pcDNA3) were cotransfected with increasingconcentration of a plasmid expressing E6 under the control of the SV40promoter (pSV2). The results obtained in the SAOS-2 line and presentedin FIG. 11 show the following properties for each of the constructs:

the activity of p53 decreases as the concentration of E6 is increased,this decrease being quite probably a reflection of the degradation ofp53 induced by E6;

the protein V-336 exhibits a lack of sensitivity to E6;

the protein V-325 appears to be capable of being slightly activated byE6. The protein V-325 is always, and in all the situations observed,more active than p53. To confirm this difference in behaviour towardsthe protein E6, the transactivating activity of the constructs V-325 andV-336 in HPV18-positive cells (HeLa), which therefore express theprotein E6, was tested and compared with that of the wild-type p53.

In this transfection experiment carried out according to the proceduredescribed above, the various constructs were placed under the control ofthe CMV promoter (pcDNA3).

The results presented in FIG. 12 show a very clear transcriptionalactivity of the two constructs V-325 and V-336, in a context where thewild-type p53 protein is not very active, again suggesting that thesetwo constructs are insensitive to E6 contrary to the wild-type protein.

To test if this absence of sensitivity to the E6 protein is thereflection of a better stability in response to the degradation inducedby this protein, an in vitro degradation experiment was carried out.

The various molecules used in this experiment were obtained bytranslation in vitro, in reticulocytes lysate, of the moleculesdescribed in Example C1 (vector pcDNA3) using the TNT CoupledReticulocyte lysate Systems kit (Promega) according to the experimentalprotocol described by the supplier for a total reaction volume of 50 μl.

For this experiment, the hybrid molecules of this invention, V-325 andV-336 as well as the wild-type p53 procein, are produced by in vitrotranslation in the presence of 44 μCi of ³⁵S-methionine (Amersham) (1175Ci/mmol) in order to generate these radioactively labelled hybridmolecules. The E6 protein (HPV18), for its part, is produced under thesame conditions but in the absence of ³⁵S-methionine.

Next, 2 μl of each of the radiolabelled products (p53, V-325 and V-336)are incubated at 30° C. with 2 μl of nonradiolabelled E6 protein and 10μl of reticulocyte lysate in a final volume of 40 μl of 25 mM Tris-HClbuffer, pH 7.5, 100 mM NaCl, 3 mM DTT. The reaction is then stopped atvarious times by removing 7.5 μl of the reaction medium and adding 7.5μl of migration buffer (Laemmli U. K., Nature, 227, 680-685, 1970) andthe samples thus prepared are subjected to electrophoresis on a 10%polyacrylamide gel in denaturing medium at 200 V according to theprotocol described above (Laemmli U. K., Nature, 227, 680-685, 1970).The gel is then dried and revealed with the aid of an instantimager(Packard instruments) which makes it possible to estimate the quantitiesof variants of the invention which were not degraded during thereaction.

The result of this experiment, presented in FIG. 13, shows clearly thatthe V-325 and V-336 variants are much more resistant than the wild-typep53 protein to degradation induced by E6, with again better propertiesfor the V-325 variant in terms of resistance to degradation. Theseresults indeed reflect the differences in sensitivity of the wild-typep53 protein and of the V-325 and V-336 variants to the E6 protein whichare observed at the level of the transcriptional activity in thepreceding experiments (FIGS. 11 and 12).

This behaviour makes these two constructs super wild-type candidateswhich are particularly advantageous for the treatment of pathologieslinked to infection with HPV16 or HPV18.

D2.3—Effect of a dominant-negative p53 mutant on the transactivatingfunction

In this experiment, the mutant H175, described as dominant-oncogenic anddominant-negative with respect to the wild-type p53 protein, was used.In this transfection experiment carried out according to the proceduredescribed above, the various constructs as well as the H175 mutant wereplaced under the control of the CMV promoter (pcDNA3). Each of theconstructs was co-transfected with increasing concentrations of theplasmid expressing the mutant H175.

The results presented in FIG. 14 show the following properties for eachof the constructs:

the p53 protein sees its transactivating activity decrease when it is inthe presence of an excess of mutated form H175, which indeed correspondsto a physiological situation since it is known that this type of mutantform is much more stable than the wild-type p53 and is therefore alwaysin excess. The dominant-negative effect of this mutant is thereforeindeed measured here.

the AS protein exhibits an increased sensitivity to thedominant-negative effect of the mutant H175, since it is sensitive tolower concentrations of the latter;

on the contrary, the proteins V-325 and V-336 are not only lesssensitive to the dominant-negative effect but even see their activityincreased in a dose-dependent manner in the presence of the mutant formH175. Again, in this test, the protein V-325 exhibits an increasedeffect compared with the protein V-336, confirming it a little more inits possible status as super wild-type.

D2.4—Effect of the hdm2 protein on the transactivating function

The protocols used are identical to those described in Example D1.1. Inthis transfection experiment, the constructs placed under the control ofthe CMV promoter (pcDNA3) are cotransfected with increasingconcentrations of a plasmid expressing hdm2 (fragment 1-134) under thecontrol of the CMV promoter (pcDNA3). The results obtained in the SAOS-2line and presented in FIG. 15 show the following properties for each ofthe constructs:

the activity of the p53 protein decreases as the concentration of hdm2increases, which indeed corresponds to a physiological situation.

the V-325 protein appears to be insensitive to this inhibition by hdm2.

This behaviour makes the V-325 protein a particularly advantageoussuperwild-type candidate for the treatment of pathologies linked to anoverexpression of hdm2, and more particularly, to the treatment ofpathologies linked to the overexpression of cellular proteins whichinteract with the N-terminal domain of the p53 protein.

The results of these four experiments show clearly that the variantsaccording to the invention, especially the variants containing theregion 75-325-lz or 75-336-lz, exhibit 1) an increased transactivatingactivity, 2) a lower sensitivity to the effect of the E6 protein fromHPV18, 3) the absence of sensitivity to the dominant-negative effect ofsome mutants of p53 and even an increase in its activity in such acontext, and 4) an absence of sensitivity to the hdm2 protein. Thesevarious properties are completely remarkable and unexpected and conferon the variants of the invention considerable therapeutic advantages.

D3—Effect on Cell Growth

The effect of the constructs V-325 and V-336 on cell growth was testedin parallel with p53 on various types of cell lines in an experiment forforming colonies resistant to neomycin following transfection withplasmids expressing these three proteins.

In this transfection experiment carried out according to the proceduredescribed above, the various constructs were placed under the control ofthe CMV promoter (pcDNA3). Procedure for formation of colonies resistantto neomycin

48 hours after transfection, the cells are scraped and transferred intoPetri dishes 10 cm in diameter and allowed to grow again with 10 ml ofDMEM medium supplemented with 10% heat-inactivated foetal calf serum andcontaining 400 μg/ml of geneticin (G418). Following a selection of 15days in the presence of G418, the number of NeOR colonies is determinedby counting after staining with fuchsin.

This experiment was carried out on various cell types in which thestatus of the p53 and Ras proteins is presented in Table 2.

TABLE 2 Status of the cell lines used in the test of formation ofNeo^(R) colonies hdm2 No. Line p53 Ras overexpression ATCC SAOS-2 -/- ?− HTB 85 HCT 116 ? mutated Ki-Ras − CCL 247 H 322 L 248 ? − (*) H 460wild-type mutated Ki-Ras − HTB 177 HeLa wild-type ? − CCL 2 OsA-Cl ? ? +(**) (*) Putnam et al., Surg. Oncol., 1 (1993), 49 (**) Oliner et al.,Nature, 358, (1992), 80

The results of these experiments are presented in Table 3 and in FIG.16.

TABLE 3 Formation of Neo^(R) colonies wild-type Line Vector p53 V-325V-336 SAOS-2 253 17 12 13 HCT116 112 62 58 61 H 322 93 5 2 3 H 460 153110 87 92 HeLa 172 151 31 47

These results show that the constructs V-325 and V-336 possess thecapacity to block cell growth in a manner which is at least as effectiveas the wild-type p53 protein in cellular situations where the latter canfunction normally (wild-type p53 or double deletant), but especiallythat they conserve this activity even in cellular situations where thewild-type p53 protein has very little activity (HeLa cells expressingthe E6 protein from HPV18 and OsA-CL cells showing overexpression of thehdm2 protein). This property confers on the variants of the inventionconsiderable therapeutic advantages.

D4—Apoptotic activity of the variants of the invention

The apoptotic activity of the variants of the invention was studiedusing the EB, EB-1 and EB-V325 cells and the induction conditionsdescribed above (Example D1).

The cells thus induced (10⁶ cells) are fixed and permeabilized by a40-minute incubation in 1 ml of Permeafix (Ortho Diagnostic SystemsInc.), then washed twice in buffer A (PBS (Gibco BRL) supplemented with0.5% Tween 20), before being resuspended and incubated for one hour atroom temperature in 100 μl of buffer A supplemented with 2% BSA(PBS-BSA) and 1 μg of the monoclonal antibody pAb240. After two newwashes in PBS-BSA buffer, the cells are incubated for 1 hour at roomtemperature in 100 μl of the same buffer supplemented with 1 μg of asecondary polyclonal antibody coupled to fluorescein (GAM-FITC(Immunotech)). Next, the cells are washed twice in buffer A, resuspendedin 1 ml of the same buffer containing 5 μg of propidium iodide and 1 mgof RNase (DNase-free), and incubated for 30 min at room temperaturebefore being analysed by flow cytometry.

The results of a 24- and 48-hour induction experiment, carried out onthe EB-1 and EB-V325 cells, are presented in FIG. 17. Under theseconditions, the cells expressing the wild-type p53 protein or itsvariant V-325 (detected by the antibody pAb240) are predominantlydistributed in the G1 and sub-G1 phase (apoptosis) after 24 hours ofinduction, then essentially in sub-G1 after 48 hours. This resultclearly indicates that the V-325 protein is capable, just like thewild-type p53 protein, of inducing apoptosis.

The results of a kinetic induction experiment carried out on the EBcells and the EB-1 and EB-V325 clones presented in FIG. 18 show that theV-325 variant appears to induce apoptosis more rapidly and moremassively than the wild-type p53 protein. Taking into account the factthat the two proteins appear to be expressed at comparable levels inthese clones (cf § D1), this result strengthens the idea of an improvedactivity of the V-325 variant relative to that of the wild-type p53protein.

59 112 base pairs nucleic acid single linear cDNA not provided 1AGATCTGAAG GCCCTCAAGG AGAAGCTGAA GGCCCTGGAG GAGAAGCTGA AGGCCCTGGA 60GGAGAAGCTG AAGGCACTAG TGGGGGAGCG ATGATGAATC GATATCGCGG CC 112 266 basepairs nucleic acid single linear cDNA not provided 2 AAGCTTGAATTCGTTAACAT GTCCACGGCC CCCCCGACCG ATGTCAGCCT GGGGGACGAG 60 CTCCACTTAGACGGCGAGGA CGTGGCGATG GCGCATGCCG ACGCGCTAGA CGATTTCGA 120 CTGGACATGTTGGGGGACGG GGATTCCCCG GGGCCGGGAT TTACCCCCCA CGACTCCGC 180 CCCTACGGCGCTCTGGATAT GGCCGACTTC GAGTTTGAGC AGATGTTTAC CGATGCCCT 240 GGAATTGACGAGTACGGTGG TCGACC 266 76 base pairs nucleic acid single linear cDNA notprovided 3 TCGAGCCTGC AGCCTAGAGC CTTCCAAGCC CTCATGAAGG AGGAAAGCCCAAACTGCTAG 60 TGAGGATCCG CGGCCG 76 788 base pairs nucleic acid singlelinear cDNA not provided 4 GGGAAGCTTG GGCCGGGTCG ACCTGCACCA GCAGCTCCTACACCGGCGGC CCCTGCACCA 60 GCCCCCTCCT GGCCCCTGTC ATCTTCTGTC CCTTCCCAGAAAACCTACCA GGGCAGCTAC 120 GGTTTCCGTC TGGGCTTCTT GCATTCTGGG ACAGCCAAGTCTGTGACTTG CACGTACTCC 180 CCTGCCCTCA ACAAGATGTT TTGCCAACTG GCCAAGACCTGCCCTGTGCA GCTGTGGGTT 240 GATTCCACAC CCCCGCCCGG CACCCGCGTC CGCGCCATGGCCATCTACAA GCAGTCACAG 300 CACATGACGG AGGTTGTGAG GCGCTGCCCC CACCATGAGCGCTGCTCAGA TAGCGATGGT 360 CTGGCCCCTC CTCAGCATCT TATCCGAGTG GAAGGAAATTTGCGTGTGGA GTATTTGGAT 420 GACAGAAACA CTTTTCGACA TAGTGTGGTG GTGCCCTATGAGCCGCCTGA GGTTGGCTCT 480 GACTGTACCA CCATCCACTA CAACTACATG TGTAACAGTTCCTGCATGGG CGGCATGAAC 540 CGGAGGCCCA TCCTCACCAT CATCACACTG GAAGACTCCAGTGGTAATCT ACTGGGACGG 600 AACAGCTTTG AGGTGCGTGT TTGTGCCTGT CCTGGGAGAGACCGGCGCAC AGAGGAAGAG 660 AATCTCCGCA AGAAAGGGGA GCCTCACCAC GAGCTGCCCCCAGGGAGCAC TAAGCGAGCA 720 CTGCCCAACA ACACCAGCTC CTCTCCCCAG CCAAAGAAGAAACCACTGGA TGGGGATCCG 780 CGGCCGCC 788 821 base pairs nucleic acidsingle linear cDNA not provided 5 GGGAAGCTTG GGCCGGGTCG ACCTGCACCAGCAGCTCCTA CACCGGCGGC CCCTGCACCA 60 GCCCCCTCCT GGCCCCTGTC ATCTTCTGTCCCTTCCCAGA AAACCTACCA GGGCAGCTAC 120 GGTTTCCGTC TGGGCTTCTT GCATTCTGGGACAGCCAAGT CTGTGACTTG CACGTACTCC 180 CCTGCCCTCA ACAAGATGTT TTGCCAACTGGCCAAGACCT GCCCTGTGCA GCTGTGGGTT 240 GATTCCACAC CCCCGCCCGG CACCCGCGTCCGCGCCATGG CCATCTACAA GCAGTCACAG 300 CACATGACGG AGGTTGTGAG GCGCTGCCCCCACCATGAGC GCTGCTCAGA TAGCGATGGT 360 CTGGCCCCTC CTCAGCATCT TATCCGAGTGGAAGGAAATT TGCGTGTGGA GTATTTGGTA 420 GACAGAAACA CTTTTCGACA TAGTGTGGTGGTGCCCTATG AGCCGCCTGA GGTTGGCTCT 480 GACTGTACCA CCATCCACTA CAACTACATGTGTAACAGTT CCTGCATGGG CGGCATGAAC 540 CGGAGGCCCA TCCTCACCAT CATCACACTGGAAGACTCCA GTGGTAATCT ACTGGGACGG 600 AACAGCTTTG AGGTGCGTGT TTGTGCCTGTCCTGGGAGAG ACCGGCGCAC AGAGGAAGAG 660 AATCTCCGCA AGAAAGGGGA GCCTCACCACGAGCTGCCCC CAGGGAGCAC TAAGCGAGCA 720 CTGCCCAACA ACACCAGCTC CTCTCCCCAGCCAAAGAAGA AACCACTGGA TGGAGAATAT 780 TTCACCCTTC AGATCCGTGG GCGTGAGGATCCGCGGCCGC C 821 15 base pairs nucleic acid single linear other nucleicacid /desc = “oligonucleotide” not provided 6 ATGGAGGAGC CGCAG 15 42base pairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” not provided 7 GGCGGCCGCG ATATCGATTC ATCAGTCTGAGTCAGGCCCT TC 42 36 base pairs nucleic acid single linear other nucleicacid /desc = “oligonucleotide” not provided 8 GGCGGCCGCG ATATCGATTCATCAGCTCGA GTGAGC 36 43 base pairs nucleic acid single linear othernucleic acid /desc = “oligonucleotide” not provided 9 TCGAGCCTGCAGCCTAGAGC CTTCCAAGCC CTCATGAAGG AGG 43 31 base pairs nucleic acidsingle linear other nucleic acid /desc = “oligonucleotide” not provided10 AAAGCCCAAA CTGCTGATGA ATCGATATCG C 31 30 base pairs nucleic acidsingle linear other nucleic acid /desc = “oligonucleotide” not provided11 TGAGGGCTTG GAAGGCTCTA GGCTGCAGGC 30 44 base pairs nucleic acid singlelinear other nucleic acid /desc = “oligonucleotide” not provided 12GGCCGCGATA TCGATTCATC AGCAGTTTGG GCTTTCCTCC TTCA 44 39 base pairsnucleic acid single linear other nucleic acid /desc = “oligonucleotide”not provided 13 GGGAAGCTTG GGCCGGGTCG ACCTGCACCA GCAGCTCCT 39 32 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” not provided 14 GGCGGCCGCG GATCCCCATC CAGTGGTTTC TT 3215 base pairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” not provided 15 ATCTGAATGG CGCTC 15 32 base pairsnucleic acid single linear other nucleic acid /desc = “oligonucleotide”not provided 16 GGCGGCCGCG GATCCTCACG CCCACGGATC TG 32 39 base pairsnucleic acid single linear other nucleic acid /desc = “oligonucleotide”not provided 17 AAGCTTGAAT TCGTTAACAT GTCCACGGCC CCCCCGACC 39 23 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” not provided 18 GGTCGACCAC CGTACTCGTC AAT 23 33 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” not provided 19 GATCTGAAGG CCCTCAAGGA GAAGCTGAAG GCC33 36 base pairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” not provided 20 CTGGAGGAGA AGCTGAAGGC CCTGGAGGAGAAGCTG 36 38 base pairs nucleic acid single linear other nucleic acid/desc = “oligonucleotide” not provided 21 AAGGCACTAG TGGGGGAGCGATGATGAATC GATATCGC 38 38 base pairs nucleic acid single linear othernucleic acid /desc = “oligonucleotide” not provided 22 AAGGCACTAGTGGGGGAGCG ATGATGAATC GATATCGC 38 36 base pairs nucleic acid singlelinear other nucleic acid /desc = “oligonucleotide” not provided 23TAGTGCCTTC AGCTTCTCCT CCAGGGCCTT CAGCTT 36 33 base pairs nucleic acidsingle linear other nucleic acid /desc = “oligonucleotide” not provided24 GGCCGCGATA TCGATTCATC ATCGCTCCCC CAC 33 1095 base pairs nucleic acidsingle linear cDNA not provided CDS 1..1089 25 ATG TCC ACG GCC CCC CCGACC GAT GTC AGC CTG GGG GAC GAG CTC CAC 48 Met Ser Thr Ala Pro Pro ThrAsp Val Ser Leu Gly Asp Glu Leu His 1 5 10 15 TTA GAC GGC GAG GAC GTGGCG ATG GCG CAT GCC GAC GCG CTA GAC GAT 96 Leu Asp Gly Glu Asp Val AlaMet Ala His Ala Asp Ala Leu Asp Asp 20 25 30 TTC GAT CTG GAC ATG TTG GGGGAC GGG GAT TCC CCG GGG CCG GGA TTT 144 Phe Asp Leu Asp Met Leu Gly AspGly Asp Ser Pro Gly Pro Gly Phe 35 40 45 ACC CCC CAC GAC TCC GCC CCC TACGGC GCT CTG GAT ATG GCC GAC TTC 192 Thr Pro His Asp Ser Ala Pro Tyr GlyAla Leu Asp Met Ala Asp Phe 50 55 60 GAG TTT GAG CAG ATG TTT ACC GAT GCCCTT GGA ATT GAC GAG TAC GGT 240 Glu Phe Glu Gln Met Phe Thr Asp Ala LeuGly Ile Asp Glu Tyr Gly 65 70 75 80 GGT CGA CCT GCA CCA GCA GCT CCT ACACCG GCG GCC CCT GCA CCA GCC 288 Gly Arg Pro Ala Pro Ala Ala Pro Thr ProAla Ala Pro Ala Pro Ala 85 90 95 CCC TCC TGG CCC CTG TCA TCT TCT GTC CCTTCC CAG AAA ACC TAC CAG 336 Pro Ser Trp Pro Leu Ser Ser Ser Val Pro SerGln Lys Thr Tyr Gln 100 105 110 GGC AGC TAC GGT TTC CGT CTG GGC TTC TTGCAT TCT GGG ACA GCC AAG 384 Gly Ser Tyr Gly Phe Arg Leu Gly Phe Leu HisSer Gly Thr Ala Lys 115 120 125 TCT GTG ACT TGC ACG TAC TCC CCT GCC CTCAAC AAG ATG TTT TGC CAA 432 Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu AsnLys Met Phe Cys Gln 130 135 140 CTG GCC AAG ACC TGC CCT GTG CAG CTG TGGGTT GAT TCC ACA CCC CCG 480 Leu Ala Lys Thr Cys Pro Val Gln Leu Trp ValAsp Ser Thr Pro Pro 145 150 155 160 CCC GGC ACC CGC GTC CGC GCC ATG GCCATC TAC AAG CAG TCA CAG CAC 528 Pro Gly Thr Arg Val Arg Ala Met Ala IleTyr Lys Gln Ser Gln His 165 170 175 ATG ACG GAG GTT GTG AGG CGC TGC CCCCAC CAT GAG CGC TGC TCA GAT 576 Met Thr Glu Val Val Arg Arg Cys Pro HisHis Glu Arg Cys Ser Asp 180 185 190 AGC GAT GGT CTG GCC CCT CCT CAG CATCTT ATC CGA GTG GAA GGA AAT 624 Ser Asp Gly Leu Ala Pro Pro Gln His LeuIle Arg Val Glu Gly Asn 195 200 205 TTG CGT GTG GAG TAT TTG GAT GAC AGAAAC ACT TTT CGA CAT AGT GTG 672 Leu Arg Val Glu Tyr Leu Asp Asp Arg AsnThr Phe Arg His Ser Val 210 215 220 GTG GTG CCC TAT GAG CCG CCT GAG GTTGGC TCT GAC TGT ACC ACC ATC 720 Val Val Pro Tyr Glu Pro Pro Glu Val GlySer Asp Cys Thr Thr Ile 225 230 235 240 CAC TAC AAC TAC ATG TGT AAC AGTTCC TGC ATG GGC GGC ATG AAC CGG 768 His Tyr Asn Tyr Met Cys Asn Ser SerCys Met Gly Gly Met Asn Arg 245 250 255 AGG CCC ATC CTC ACC ATC ATC ACACTG GAA GAC TCC AGT GGT AAT CTA 816 Arg Pro Ile Leu Thr Ile Ile Thr LeuGlu Asp Ser Ser Gly Asn Leu 260 265 270 CTG GGA CGG AAC AGC TTT GAG GTGCGT GTT TGT GCC TGT CCT GGG AGA 864 Leu Gly Arg Asn Ser Phe Glu Val ArgVal Cys Ala Cys Pro Gly Arg 275 280 285 GAC CGG CGC ACA GAG GAA GAG AATCTC CGC AAG AAA GGG GAG CCT CAC 912 Asp Arg Arg Thr Glu Glu Glu Asn LeuArg Lys Lys Gly Glu Pro His 290 295 300 CAC GAG CTG CCC CCA GGG AGC ACTAAG CGA GCA CTG CCC AAC AAC ACC 960 His Glu Leu Pro Pro Gly Ser Thr LysArg Ala Leu Pro Asn Asn Thr 305 310 315 320 AGC TCC TCT CCC CAG CCA AAGAAG AAA CCA CTG GAT GGG GAT CTG AAG 1008 Ser Ser Ser Pro Gln Pro Lys LysLys Pro Leu Asp Gly Asp Leu Lys 325 330 335 GCC CTC AAG GAG AAG CTG AAGGCC CTG GAG GAG AAG CTG AAG GCC CTG 1056 Ala Leu Lys Glu Lys Leu Lys AlaLeu Glu Glu Lys Leu Lys Ala Leu 340 345 350 GAG GAG AAG CTG AAG GCA CTAGTG GGG GAG CGA TGATGA 1095 Glu Glu Lys Leu Lys Ala Leu Val Gly Glu Arg355 360 363 amino acids amino acid linear protein not provided 26 MetSer Thr Ala Pro Pro Thr Asp Val Ser Leu Gly Asp Glu Leu His 1 5 10 15Leu Asp Gly Glu Asp Val Ala Met Ala His Ala Asp Ala Leu Asp Asp 20 25 30Phe Asp Leu Asp Met Leu Gly Asp Gly Asp Ser Pro Gly Pro Gly Phe 35 40 45Thr Pro His Asp Ser Ala Pro Tyr Gly Ala Leu Asp Met Ala Asp Phe 50 55 60Glu Phe Glu Gln Met Phe Thr Asp Ala Leu Gly Ile Asp Glu Tyr Gly 65 70 7580 Gly Arg Pro Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala 85 9095 Pro Ser Trp Pro Leu Ser Ser Ser Val Pro Ser Gln Lys Thr Tyr Gln 100105 110 Gly Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys115 120 125 Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe CysGln 130 135 140 Leu Ala Lys Thr Cys Pro Val Gln Leu Trp Val Asp Ser ThrPro Pro 145 150 155 160 Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr LysGln Ser Gln His 165 170 175 Met Thr Glu Val Val Arg Arg Cys Pro His HisGlu Arg Cys Ser Asp 180 185 190 Ser Asp Gly Leu Ala Pro Pro Gln His LeuIle Arg Val Glu Gly Asn 195 200 205 Leu Arg Val Glu Tyr Leu Asp Asp ArgAsn Thr Phe Arg His Ser Val 210 215 220 Val Val Pro Tyr Glu Pro Pro GluVal Gly Ser Asp Cys Thr Thr Ile 225 230 235 240 His Tyr Asn Tyr Met CysAsn Ser Ser Cys Met Gly Gly Met Asn Arg 245 250 255 Arg Pro Ile Leu ThrIle Ile Thr Leu Glu Asp Ser Ser Gly Asn Leu 260 265 270 Leu Gly Arg AsnSer Phe Glu Val Arg Val Cys Ala Cys Pro Gly Arg 275 280 285 Asp Arg ArgThr Glu Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His 290 295 300 His GluLeu Pro Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr 305 310 315 320Ser Ser Ser Pro Gln Pro Lys Lys Lys Pro Leu Asp Gly Asp Leu Lys 325 330335 Ala Leu Lys Glu Lys Leu Lys Ala Leu Glu Glu Lys Leu Lys Ala Leu 340345 350 Glu Glu Lys Leu Lys Ala Leu Val Gly Glu Arg 355 360 1128 basepairs nucleic acid single linear cDNA not provided CDS 1..1122 27 ATGTCC ACG GCC CCC CCG ACC GAT GTC AGC CTG GGG GAC GAG CTC CAC 48 Met SerThr Ala Pro Pro Thr Asp Val Ser Leu Gly Asp Glu Leu His 1 5 10 15 TTAGAC GGC GAG GAC GTG GCG ATG GCG CAT GCC GAC GCG CTA GAC GAT 96 Leu AspGly Glu Asp Val Ala Met Ala His Ala Asp Ala Leu Asp Asp 20 25 30 TTC GATCTG GAC ATG TTG GGG GAC GGG GAT TCC CCG GGG CCG GGA TTT 144 Phe Asp LeuAsp Met Leu Gly Asp Gly Asp Ser Pro Gly Pro Gly Phe 35 40 45 ACC CCC CACGAC TCC GCC CCC TAC GGC GCT CTG GAT ATG GCC GAC TTC 192 Thr Pro His AspSer Ala Pro Tyr Gly Ala Leu Asp Met Ala Asp Phe 50 55 60 GAG TTT GAG CAGATG TTT ACC GAT GCC CTT GGA ATT GAC GAG TAC GGT 240 Glu Phe Glu Gln MetPhe Thr Asp Ala Leu Gly Ile Asp Glu Tyr Gly 65 70 75 80 GGT CGA CCT GCACCA GCA GCT CCT ACA CCG GCG GCC CCT GCA CCA GCC 288 Gly Arg Pro Ala ProAla Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala 85 90 95 CCC TCC TGG CCC CTGTCA TCT TCT GTC CCT TCC CAG AAA ACC TAC CAG 336 Pro Ser Trp Pro Leu SerSer Ser Val Pro Ser Gln Lys Thr Tyr Gln 100 105 110 GGC AGC TAC GGT TTCCGT CTG GGC TTC TTG CAT TCT GGG ACA GCC AAG 384 Gly Ser Tyr Gly Phe ArgLeu Gly Phe Leu His Ser Gly Thr Ala Lys 115 120 125 TCT GTG ACT TGC ACGTAC TCC CCT GCC CTC AAC AAG ATG TTT TGC CAA 432 Ser Val Thr Cys Thr TyrSer Pro Ala Leu Asn Lys Met Phe Cys Gln 130 135 140 CTG GCC AAG ACC TGCCCT GTG CAG CTG TGG GTT GAT TCC ACA CCC CCG 480 Leu Ala Lys Thr Cys ProVal Gln Leu Trp Val Asp Ser Thr Pro Pro 145 150 155 160 CCC GGC ACC CGCGTC CGC GCC ATG GCC ATC TAC AAG CAG TCA CAG CAC 528 Pro Gly Thr Arg ValArg Ala Met Ala Ile Tyr Lys Gln Ser Gln His 165 170 175 ATG ACG GAG GTTGTG AGG CGC TGC CCC CAC CAT GAG CGC TGC TCA GAT 576 Met Thr Glu Val ValArg Arg Cys Pro His His Glu Arg Cys Ser Asp 180 185 190 AGC GAT GGT CTGGCC CCT CCT CAG CAT CTT ATC CGA GTG GAA GGA AAT 624 Ser Asp Gly Leu AlaPro Pro Gln His Leu Ile Arg Val Glu Gly Asn 195 200 205 TTG CGT GTG GAGTAT TTG GAT GAC AGA AAC ACT TTT CGA CAT AGT GTG 672 Leu Arg Val Glu TyrLeu Asp Asp Arg Asn Thr Phe Arg His Ser Val 210 215 220 GTG GTG CCC TATGAG CCG CCT GAG GTT GGC TCT GAC TGT ACC ACC ATC 720 Val Val Pro Tyr GluPro Pro Glu Val Gly Ser Asp Cys Thr Thr Ile 225 230 235 240 CAC TAC AACTAC ATG TGT AAC AGT TCC TGC ATG GGC GGC ATG AAC CGG 768 His Tyr Asn TyrMet Cys Asn Ser Ser Cys Met Gly Gly Met Asn Arg 245 250 255 AGG CCC ATCCTC ACC ATC ATC ACA CTG GAA GAC TCC AGT GGT AAT CTA 816 Arg Pro Ile LeuThr Ile Ile Thr Leu Glu Asp Ser Ser Gly Asn Leu 260 265 270 CTG GGA CGGAAC AGC TTT GAG GTG CGT GTT TGT GCC TGT CCT GGG AGA 864 Leu Gly Arg AsnSer Phe Glu Val Arg Val Cys Ala Cys Pro Gly Arg 275 280 285 GAC CGG CGCACA GAG GAA GAG AAT CTC CGC AAG AAA GGG GAG CCT CAC 912 Asp Arg Arg ThrGlu Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His 290 295 300 CAC GAG CTGCCC CCA GGG AGC ACT AAG CGA GCA CTG CCC AAC AAC ACC 960 His Glu Leu ProPro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr 305 310 315 320 AGC TCCTCT CCC CAG CCA AAG AAG AAA CCA CTG GAT GGA GAA TAT TTC 1008 Ser Ser SerPro Gln Pro Lys Lys Lys Pro Leu Asp Gly Glu Tyr Phe 325 330 335 ACC CTTCAG ATC CGT GGG CGT GAG GAT CTG AAG GCC CTC AAG GAG AAG 1056 Thr Leu GlnIle Arg Gly Arg Glu Asp Leu Lys Ala Leu Lys Glu Lys 340 345 350 CTG AAGGCC CTG GAG GAG AAG CTG AAG GCC CTG GAG GAG AAG CTG AAG 1104 Leu Lys AlaLeu Glu Glu Lys Leu Lys Ala Leu Glu Glu Lys Leu Lys 355 360 365 GCA CTAGTG GGG GAG CGA TGATGA 1128 Ala Leu Val Gly Glu Arg 370 374 amino acidsamino acid linear protein not provided 28 Met Ser Thr Ala Pro Pro ThrAsp Val Ser Leu Gly Asp Glu Leu His 1 5 10 15 Leu Asp Gly Glu Asp ValAla Met Ala His Ala Asp Ala Leu Asp Asp 20 25 30 Phe Asp Leu Asp Met LeuGly Asp Gly Asp Ser Pro Gly Pro Gly Phe 35 40 45 Thr Pro His Asp Ser AlaPro Tyr Gly Ala Leu Asp Met Ala Asp Phe 50 55 60 Glu Phe Glu Gln Met PheThr Asp Ala Leu Gly Ile Asp Glu Tyr Gly 65 70 75 80 Gly Arg Pro Ala ProAla Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala 85 90 95 Pro Ser Trp Pro LeuSer Ser Ser Val Pro Ser Gln Lys Thr Tyr Gln 100 105 110 Gly Ser Tyr GlyPhe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys 115 120 125 Ser Val ThrCys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys Gln 130 135 140 Leu AlaLys Thr Cys Pro Val Gln Leu Trp Val Asp Ser Thr Pro Pro 145 150 155 160Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gln Ser Gln His 165 170175 Met Thr Glu Val Val Arg Arg Cys Pro His His Glu Arg Cys Ser Asp 180185 190 Ser Asp Gly Leu Ala Pro Pro Gln His Leu Ile Arg Val Glu Gly Asn195 200 205 Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn Thr Phe Arg His SerVal 210 215 220 Val Val Pro Tyr Glu Pro Pro Glu Val Gly Ser Asp Cys ThrThr Ile 225 230 235 240 His Tyr Asn Tyr Met Cys Asn Ser Ser Cys Met GlyGly Met Asn Arg 245 250 255 Arg Pro Ile Leu Thr Ile Ile Thr Leu Glu AspSer Ser Gly Asn Leu 260 265 270 Leu Gly Arg Asn Ser Phe Glu Val Arg ValCys Ala Cys Pro Gly Arg 275 280 285 Asp Arg Arg Thr Glu Glu Glu Asn LeuArg Lys Lys Gly Glu Pro His 290 295 300 His Glu Leu Pro Pro Gly Ser ThrLys Arg Ala Leu Pro Asn Asn Thr 305 310 315 320 Ser Ser Ser Pro Gln ProLys Lys Lys Pro Leu Asp Gly Glu Tyr Phe 325 330 335 Thr Leu Gln Ile ArgGly Arg Glu Asp Leu Lys Ala Leu Lys Glu Lys 340 345 350 Leu Lys Ala LeuGlu Glu Lys Leu Lys Ala Leu Glu Glu Lys Leu Lys 355 360 365 Ala Leu ValGly Glu Arg 370 765 base pairs nucleic acid single linear cDNA notprovided CDS 1..759 29 ATG TCC ACG GCC CCC CCG ACC GAT GTC AGC CTG GGGGAC GAG CTC CAC 48 Met Ser Thr Ala Pro Pro Thr Asp Val Ser Leu Gly AspGlu Leu His 1 5 10 15 TTA GAC GGC GAG GAC GTG GCG ATG GCG CAT GCC GACGCG CTA GAC GAT 96 Leu Asp Gly Glu Asp Val Ala Met Ala His Ala Asp AlaLeu Asp Asp 20 25 30 TTC GAT CTG GAC ATG TTG GGG GAC GGG GAT TCC CCG GGGCCG GGA TTT 144 Phe Asp Leu Asp Met Leu Gly Asp Gly Asp Ser Pro Gly ProGly Phe 35 40 45 ACC CCC CAC GAC TCC GCC CCC TAC GGC GCT CTG GAT ATG GCCGAC TTC 192 Thr Pro His Asp Ser Ala Pro Tyr Gly Ala Leu Asp Met Ala AspPhe 50 55 60 GAG TTT GAG CAG ATG TTT ACC GAT GCC CTT GGA ATT GAC GAG TACGGT 240 Glu Phe Glu Gln Met Phe Thr Asp Ala Leu Gly Ile Asp Glu Tyr Gly65 70 75 80 GGT CGA CCT GCA CCA GCA GCT CCT ACA CCG GCG GCC CCT GCA CCAGCC 288 Gly Arg Pro Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala85 90 95 CCC TCC TGG CCC CTG TCA TCT TCT GTC CCT TCC CAG AAA ACC TAC CAG336 Pro Ser Trp Pro Leu Ser Ser Ser Val Pro Ser Gln Lys Thr Tyr Gln 100105 110 GGC AGC TAC GGT TTC CGT CTG GGC TTC TTG CAT TCT GGG ACA GCC AAG384 Gly Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys 115120 125 TCT GTG ACT TGC ACG TAC TCC CCT GCC CTC AAC AAG ATG TTT TGC CAA432 Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys Gln 130135 140 CTG GCC AAG ACC TGC CCT GTG CAG CTG TGG GTT GAT TCC ACA CCC CCG480 Leu Ala Lys Thr Cys Pro Val Gln Leu Trp Val Asp Ser Thr Pro Pro 145150 155 160 CCC GGC ACC CGC GTC CGC GCC ATG GCC ATC TAC AAG CAG TCA CAGCAC 528 Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gln Ser Gln His165 170 175 ATG ACG GAG GTT GTG AGG CGC TGC CCC CAC CAT GAG CGC TGC TCAGAT 576 Met Thr Glu Val Val Arg Arg Cys Pro His His Glu Arg Cys Ser Asp180 185 190 AGC GAT GGT CTG GCC CCT CCT CAG CAT CTT ATC CGA GTG GAA GGAAAT 624 Ser Asp Gly Leu Ala Pro Pro Gln His Leu Ile Arg Val Glu Gly Asn195 200 205 TTG CGT GTG GAG TAT TTC ACC CTT CAG ATC CGT GGG CGT GAG CGCTTC 672 Leu Arg Val Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu Arg Phe210 215 220 GAG ATG TTC CGA GAG CTG AAT GAG GCC TTG GAA CTC AAG GAT GCCCAG 720 Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp Ala Gln225 230 235 240 GCT GGG AAG GAG CCA GGG GGG AGC AGG GCT CAC TCG AGCTGATGA 765 Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser 245 250253 amino acids amino acid linear protein not provided 30 Met Ser ThrAla Pro Pro Thr Asp Val Ser Leu Gly Asp Glu Leu His 1 5 10 15 Leu AspGly Glu Asp Val Ala Met Ala His Ala Asp Ala Leu Asp Asp 20 25 30 Phe AspLeu Asp Met Leu Gly Asp Gly Asp Ser Pro Gly Pro Gly Phe 35 40 45 Thr ProHis Asp Ser Ala Pro Tyr Gly Ala Leu Asp Met Ala Asp Phe 50 55 60 Glu PheGlu Gln Met Phe Thr Asp Ala Leu Gly Ile Asp Glu Tyr Gly 65 70 75 80 GlyArg Pro Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala 85 90 95 ProSer Trp Pro Leu Ser Ser Ser Val Pro Ser Gln Lys Thr Tyr Gln 100 105 110Gly Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys 115 120125 Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys Gln 130135 140 Leu Ala Lys Thr Cys Pro Val Gln Leu Trp Val Asp Ser Thr Pro Pro145 150 155 160 Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gln SerGln His 165 170 175 Met Thr Glu Val Val Arg Arg Cys Pro His His Glu ArgCys Ser Asp 180 185 190 Ser Asp Gly Leu Ala Pro Pro Gln His Leu Ile ArgVal Glu Gly Asn 195 200 205 Leu Arg Val Glu Tyr Phe Thr Leu Gln Ile ArgGly Arg Glu Arg Phe 210 215 220 Glu Met Phe Arg Glu Leu Asn Glu Ala LeuGlu Leu Lys Asp Ala Gln 225 230 235 240 Ala Gly Lys Glu Pro Gly Gly SerArg Ala His Ser Ser 245 250 816 base pairs nucleic acid single linearcDNA not provided CDS 1..810 31 ATG TCC ACG GCC CCC CCG ACC GAT GTC AGCCTG GGG GAC GAG CTC CAC 48 Met Ser Thr Ala Pro Pro Thr Asp Val Ser LeuGly Asp Glu Leu His 1 5 10 15 TTA GAC GGC GAG GAC GTG GCG ATG GCG CATGCC GAC GCG CTA GAC GAT 96 Leu Asp Gly Glu Asp Val Ala Met Ala His AlaAsp Ala Leu Asp Asp 20 25 30 TTC GAT CTG GAC ATG TTG GGG GAC GGG GAT TCCCCG GGG CCG GGA TTT 144 Phe Asp Leu Asp Met Leu Gly Asp Gly Asp Ser ProGly Pro Gly Phe 35 40 45 ACC CCC CAC GAC TCC GCC CCC TAC GGC GCT CTG GATATG GCC GAC TTC 192 Thr Pro His Asp Ser Ala Pro Tyr Gly Ala Leu Asp MetAla Asp Phe 50 55 60 GAG TTT GAG CAG ATG TTT ACC GAT GCC CTT GGA ATT GACGAG TAC GGT 240 Glu Phe Glu Gln Met Phe Thr Asp Ala Leu Gly Ile Asp GluTyr Gly 65 70 75 80 GGT CGA CCT GCA CCA GCA GCT CCT ACA CCG GCG GCC CCTGCA CCA GCC 288 Gly Arg Pro Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro AlaPro Ala 85 90 95 CCC TCC TGG CCC CTG TCA TCT TCT GTC CCT TCC CAG AAA ACCTAC CAG 336 Pro Ser Trp Pro Leu Ser Ser Ser Val Pro Ser Gln Lys Thr TyrGln 100 105 110 GGC AGC TAC GGT TTC CGT CTG GGC TTC TTG CAT TCT GGG ACAGCC AAG 384 Gly Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr AlaLys 115 120 125 TCT GTG ACT TGC ACG TAC TCC CCT GCC CTC AAC AAG ATG TTTTGC CAA 432 Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe CysGln 130 135 140 CTG GCC AAG ACC TGC CCT GTG CAG CTG TGG GTT GAT TCC ACACCC CCG 480 Leu Ala Lys Thr Cys Pro Val Gln Leu Trp Val Asp Ser Thr ProPro 145 150 155 160 CCC GGC ACC CGC GTC CGC GCC ATG GCC ATC TAC AAG CAGTCA CAG CAC 528 Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gln SerGln His 165 170 175 ATG ACG GAG GTT GTG AGG CGC TGC CCC CAC CAT GAG CGCTGC TCA GAT 576 Met Thr Glu Val Val Arg Arg Cys Pro His His Glu Arg CysSer Asp 180 185 190 AGC GAT GGT CTG GCC CCT CCT CAG CAT CTT ATC CGA GTGGAA GGA AAT 624 Ser Asp Gly Leu Ala Pro Pro Gln His Leu Ile Arg Val GluGly Asn 195 200 205 TTG CGT GTG GAG TAT TTC ACC CTT CAG ATC CGT GGG CGTGAG CGC TTC 672 Leu Arg Val Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg GluArg Phe 210 215 220 GAG ATG TTC CGA GAG CTG AAT GAG GCC TTG GAA CTC AAGGAT GCC CAG 720 Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys AspAla Gln 225 230 235 240 GCT GGG AAG GAG CCA GGG GGG AGC AGG GCT CAC TCGAGC CTG CAG CCT 768 Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser SerLeu Gln Pro 245 250 255 AGA GCC TTC CAA GCC CTC ATG AAG GAG GAA AGC CCAAAG TGC 810 Arg Ala Phe Gln Ala Leu Met Lys Glu Glu Ser Pro Lys Cys 260265 270 TGATGA 816 270 amino acids amino acid linear protein notprovided 32 Met Ser Thr Ala Pro Pro Thr Asp Val Ser Leu Gly Asp Glu LeuHis 1 5 10 15 Leu Asp Gly Glu Asp Val Ala Met Ala His Ala Asp Ala LeuAsp Asp 20 25 30 Phe Asp Leu Asp Met Leu Gly Asp Gly Asp Ser Pro Gly ProGly Phe 35 40 45 Thr Pro His Asp Ser Ala Pro Tyr Gly Ala Leu Asp Met AlaAsp Phe 50 55 60 Glu Phe Glu Gln Met Phe Thr Asp Ala Leu Gly Ile Asp GluTyr Gly 65 70 75 80 Gly Arg Pro Ala Pro Ala Ala Pro Thr Pro Ala Ala ProAla Pro Ala 85 90 95 Pro Ser Trp Pro Leu Ser Ser Ser Val Pro Ser Gln LysThr Tyr Gln 100 105 110 Gly Ser Tyr Gly Phe Arg Leu Gly Phe Leu His SerGly Thr Ala Lys 115 120 125 Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu AsnLys Met Phe Cys Gln 130 135 140 Leu Ala Lys Thr Cys Pro Val Gln Leu TrpVal Asp Ser Thr Pro Pro 145 150 155 160 Pro Gly Thr Arg Val Arg Ala MetAla Ile Tyr Lys Gln Ser Gln His 165 170 175 Met Thr Glu Val Val Arg ArgCys Pro His His Glu Arg Cys Ser Asp 180 185 190 Ser Asp Gly Leu Ala ProPro Gln His Leu Ile Arg Val Glu Gly Asn 195 200 205 Leu Arg Val Glu TyrPhe Thr Leu Gln Ile Arg Gly Arg Glu Arg Phe 210 215 220 Glu Met Phe ArgGlu Leu Asn Glu Ala Leu Glu Leu Lys Asp Ala Gln 225 230 235 240 Ala GlyLys Glu Pro Gly Gly Ser Arg Ala His Ser Ser Leu Gln Pro 245 250 255 ArgAla Phe Gln Ala Leu Met Lys Glu Glu Ser Pro Lys Cys 260 265 270 1209base pairs nucleic acid single linear cDNA not provided CDS 1..1203 33ATG TCC ACG GCC CCC CCG ACC GAT GTC AGC CTG GGG GAC GAG CTC CAC 48 MetSer Thr Ala Pro Pro Thr Asp Val Ser Leu Gly Asp Glu Leu His 1 5 10 15TTA GAC GGC GAG GAC GTG GCG ATG GCG CAT GCC GAC GCG CTA GAC GAT 96 LeuAsp Gly Glu Asp Val Ala Met Ala His Ala Asp Ala Leu Asp Asp 20 25 30 TTCGAT CTG GAC ATG TTG GGG GAC GGG GAT TCC CCG GGG CCG GGA TTT 144 Phe AspLeu Asp Met Leu Gly Asp Gly Asp Ser Pro Gly Pro Gly Phe 35 40 45 ACC CCCCAC GAC TCC GCC CCC TAC GGC GCT CTG GAT ATG GCC GAC TTC 192 Thr Pro HisAsp Ser Ala Pro Tyr Gly Ala Leu Asp Met Ala Asp Phe 50 55 60 GAG TTT GAGCAG ATG TTT ACC GAT GCC CTT GGA ATT GAC GAG TAC GGT 240 Glu Phe Glu GlnMet Phe Thr Asp Ala Leu Gly Ile Asp Glu Tyr Gly 65 70 75 80 GGT CGA CCTGCA CCA GCA GCT CCT ACA CCG GCG GCC CCT GCA CCA GCC 288 Gly Arg Pro AlaPro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala 85 90 95 CCC TCC TGG CCCCTG TCA TCT TCT GTC CCT TCC CAG AAA ACC TAC CAG 336 Pro Ser Trp Pro LeuSer Ser Ser Val Pro Ser Gln Lys Thr Tyr Gln 100 105 110 GGC AGC TAC GGTTTC CGT CTG GGC TTC TTG CAT TCT GGG ACA GCC AAG 384 Gly Ser Tyr Gly PheArg Leu Gly Phe Leu His Ser Gly Thr Ala Lys 115 120 125 TCT GTG ACT TGCACG TAC TCC CCT GCC CTC AAC AAG ATG TTT TGC CAA 432 Ser Val Thr Cys ThrTyr Ser Pro Ala Leu Asn Lys Met Phe Cys Gln 130 135 140 CTG GCC AAG ACCTGC CCT GTG CAG CTG TGG GTT GAT TCC ACA CCC CCG 480 Leu Ala Lys Thr CysPro Val Gln Leu Trp Val Asp Ser Thr Pro Pro 145 150 155 160 CCC GGC ACCCGC GTC CGC GCC ATG GCC ATC TAC AAG CAG TCA CAG CAC 528 Pro Gly Thr ArgVal Arg Ala Met Ala Ile Tyr Lys Gln Ser Gln His 165 170 175 ATG ACG GAGGTT GTG AGG CGC TGC CCC CAC CAT GAG CGC TGC TCA GAT 576 Met Thr Glu ValVal Arg Arg Cys Pro His His Glu Arg Cys Ser Asp 180 185 190 AGC GAT GGTCTG GCC CCT CCT CAG CAT CTT ATC CGA GTG GAA GGA AAT 624 Ser Asp Gly LeuAla Pro Pro Gln His Leu Ile Arg Val Glu Gly Asn 195 200 205 TTG CGT GTGGAG TAT TTG GAT GAC AGA AAC ACT TTT CGA CAT AGT GTG 672 Leu Arg Val GluTyr Leu Asp Asp Arg Asn Thr Phe Arg His Ser Val 210 215 220 GTG GTG CCCTAT GAG CCG CCT GAG GTT GGC TCT GAC TGT ACC ACC ATC 720 Val Val Pro TyrGlu Pro Pro Glu Val Gly Ser Asp Cys Thr Thr Ile 225 230 235 240 CAC TACAAC TAC ATG TGT AAC AGT TCC TGC ATG GGC GGC ATG AAC CGG 768 His Tyr AsnTyr Met Cys Asn Ser Ser Cys Met Gly Gly Met Asn Arg 245 250 255 AGG CCCATC CTC ACC ATC ATC ACA CTG GAA GAC TCC AGT GGT AAT CTA 816 Arg Pro IleLeu Thr Ile Ile Thr Leu Glu Asp Ser Ser Gly Asn Leu 260 265 270 CTG GGACGG AAC AGC TTT GAG GTG CGT GTT TGT GCC TGT CCT GGG AGA 864 Leu Gly ArgAsn Ser Phe Glu Val Arg Val Cys Ala Cys Pro Gly Arg 275 280 285 GAC CGGCGC ACA GAG GAA GAG AAT CTC CGC AAG AAA GGG GAG CCT CAC 912 Asp Arg ArgThr Glu Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His 290 295 300 CAC GAGCTG CCC CCA GGG AGC ACT AAG CGA GCA CTG CCC AAC AAC ACC 960 His Glu LeuPro Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr 305 310 315 320 AGCTCC TCT CCC CAG CCA AAG AAG AAA CCA CTG GAT GGA GAA TAT TTC 1008 Ser SerSer Pro Gln Pro Lys Lys Lys Pro Leu Asp Gly Glu Tyr Phe 325 330 335 ACCCTT CAG ATC CGT GGG CGT GAG CGC TTC GAG ATG TTC CGA GAG CTG 1056 Thr LeuGln Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu 340 345 350 AATGAG GCC TTG GAA CTC AAG GAT GCC CAG GCT GGG AAG GAG CCA GGG 1104 Asn GluAla Leu Glu Leu Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly 355 360 365 GGGAGC AGG GCT CAC TCC AGC CAC CTG AAG TCC AAA AAG GGT CAG TCT 1152 Gly SerArg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser 370 375 380 ACCTCC CGC CAT AAA AAA CTC ATG TTC AAG ACA GAA GGG CCT GAC TCA 1200 Thr SerArg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro Asp Ser 385 390 395 400GAC TGATGA 1209 Asp 401 amino acids amino acid linear protein notprovided 34 Met Ser Thr Ala Pro Pro Thr Asp Val Ser Leu Gly Asp Glu LeuHis 1 5 10 15 Leu Asp Gly Glu Asp Val Ala Met Ala His Ala Asp Ala LeuAsp Asp 20 25 30 Phe Asp Leu Asp Met Leu Gly Asp Gly Asp Ser Pro Gly ProGly Phe 35 40 45 Thr Pro His Asp Ser Ala Pro Tyr Gly Ala Leu Asp Met AlaAsp Phe 50 55 60 Glu Phe Glu Gln Met Phe Thr Asp Ala Leu Gly Ile Asp GluTyr Gly 65 70 75 80 Gly Arg Pro Ala Pro Ala Ala Pro Thr Pro Ala Ala ProAla Pro Ala 85 90 95 Pro Ser Trp Pro Leu Ser Ser Ser Val Pro Ser Gln LysThr Tyr Gln 100 105 110 Gly Ser Tyr Gly Phe Arg Leu Gly Phe Leu His SerGly Thr Ala Lys 115 120 125 Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu AsnLys Met Phe Cys Gln 130 135 140 Leu Ala Lys Thr Cys Pro Val Gln Leu TrpVal Asp Ser Thr Pro Pro 145 150 155 160 Pro Gly Thr Arg Val Arg Ala MetAla Ile Tyr Lys Gln Ser Gln His 165 170 175 Met Thr Glu Val Val Arg ArgCys Pro His His Glu Arg Cys Ser Asp 180 185 190 Ser Asp Gly Leu Ala ProPro Gln His Leu Ile Arg Val Glu Gly Asn 195 200 205 Leu Arg Val Glu TyrLeu Asp Asp Arg Asn Thr Phe Arg His Ser Val 210 215 220 Val Val Pro TyrGlu Pro Pro Glu Val Gly Ser Asp Cys Thr Thr Ile 225 230 235 240 His TyrAsn Tyr Met Cys Asn Ser Ser Cys Met Gly Gly Met Asn Arg 245 250 255 ArgPro Ile Leu Thr Ile Ile Thr Leu Glu Asp Ser Ser Gly Asn Leu 260 265 270Leu Gly Arg Asn Ser Phe Glu Val Arg Val Cys Ala Cys Pro Gly Arg 275 280285 Asp Arg Arg Thr Glu Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His 290295 300 His Glu Leu Pro Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr305 310 315 320 Ser Ser Ser Pro Gln Pro Lys Lys Lys Pro Leu Asp Gly GluTyr Phe 325 330 335 Thr Leu Gln Ile Arg Gly Arg Glu Arg Phe Glu Met PheArg Glu Leu 340 345 350 Asn Glu Ala Leu Glu Leu Lys Asp Ala Gln Ala GlyLys Glu Pro Gly 355 360 365 Gly Ser Arg Ala His Ser Ser His Leu Lys SerLys Lys Gly Gln Ser 370 375 380 Thr Ser Arg His Lys Lys Leu Met Phe LysThr Glu Gly Pro Asp Ser 385 390 395 400 Asp 1149 base pairs nucleic acidsingle linear cDNA not provided CDS 1..1143 35 ATG GCC ACG GCC CCC CCGACC GAT GTC AGC CTG GGG GAC GAG CTC CAC 48 Met Ala Thr Ala Pro Pro ThrAsp Val Ser Leu Gly Asp Glu Leu His 1 5 10 15 TTA GAC GGC GAG GAC GTGGCG ATG GCG CAT GCC GAC GCG CTA GAC GAT 96 Leu Asp Gly Glu Asp Val AlaMet Ala His Ala Asp Ala Leu Asp Asp 20 25 30 TTC GAT CTG GAC ATG TTG GGGGAC GGG GAT TCC CCG GGG CCG GGA TTT 144 Phe Asp Leu Asp Met Leu Gly AspGly Asp Ser Pro Gly Pro Gly Phe 35 40 45 ACC CCC CAC GAC TCC GCC CCC TACGGC GCT CTG GAT ATG GCC GAC TTC 192 Thr Pro His Asp Ser Ala Pro Tyr GlyAla Leu Asp Met Ala Asp Phe 50 55 60 GAG TTT GAG CAG ATG TTT ACC GAT GCCCTT GGA ATT GAC GAG TAC GGT 240 Glu Phe Glu Gln Met Phe Thr Asp Ala LeuGly Ile Asp Glu Tyr Gly 65 70 75 80 GGT CGA CCT GCA CCA GCA GCT CCT ACACCG GCG GCC CCT GCA CCA GCC 288 Gly Arg Pro Ala Pro Ala Ala Pro Thr ProAla Ala Pro Ala Pro Ala 85 90 95 CCC TCC TGG CCC CTG TCA TCT TCT GTC CCTTCC CAG AAA ACC TAC CAG 336 Pro Ser Trp Pro Leu Ser Ser Ser Val Pro SerGln Lys Thr Tyr Gln 100 105 110 GGC AGC TAC GGT TTC CGT CTG GGC TTC TTGCAT TCT GGG ACA GCC AAG 384 Gly Ser Tyr Gly Phe Arg Leu Gly Phe Leu HisSer Gly Thr Ala Lys 115 120 125 TCT GTG ACT TGC ACG TAC TCC CCT GCC CTCAAC AAG ATG TTT TGC CAA 432 Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu AsnLys Met Phe Cys Gln 130 135 140 CTG GCC AAG ACC TGC CCT GTG CAG CTG TGGGTT GAT TCC ACA CCC CCG 480 Leu Ala Lys Thr Cys Pro Val Gln Leu Trp ValAsp Ser Thr Pro Pro 145 150 155 160 CCC GGC ACC CGC GTC CGC GCC ATG GCCATC TAC AAG CAG TCA CAG CAC 528 Pro Gly Thr Arg Val Arg Ala Met Ala IleTyr Lys Gln Ser Gln His 165 170 175 ATG ACG GAG GTT GTG AGG CGC TGC CCCCAC CAT GAG CGC TGC TCA GAT 576 Met Thr Glu Val Val Arg Arg Cys Pro HisHis Glu Arg Cys Ser Asp 180 185 190 AGC GAT GGT CTG GCC CCT CCT CAG CATCTT ATC CGA GTG GAA GGA AAT 624 Ser Asp Gly Leu Ala Pro Pro Gln His LeuIle Arg Val Glu Gly Asn 195 200 205 TTG CGT GTG GAG TAT TTG GAT GAC AGAAAC ACT TTT CGA CAT AGT GTG 672 Leu Arg Val Glu Tyr Leu Asp Asp Arg AsnThr Phe Arg His Ser Val 210 215 220 GTG GTG CCC TAT GAG CCG CCT GAG GTTGGC TCT GAC TGT ACC ACC ATC 720 Val Val Pro Tyr Glu Pro Pro Glu Val GlySer Asp Cys Thr Thr Ile 225 230 235 240 CAC TAC AAC TAC ATG TGT AAC AGTTCC TGC ATG GGC GGC ATG AAC CGG 768 His Tyr Asn Tyr Met Cys Asn Ser SerCys Met Gly Gly Met Asn Arg 245 250 255 AGG CCC ATC CTC ACC ATC ATC ACACTG GAA GAC TCC AGT GGT AAT CTA 816 Arg Pro Ile Leu Thr Ile Ile Thr LeuGlu Asp Ser Ser Gly Asn Leu 260 265 270 CTG GGA CGG AAC AGC TTT GAG GTGCGT GTT TGT GCC TGT CCT GGG AGA 864 Leu Gly Arg Asn Ser Phe Glu Val ArgVal Cys Ala Cys Pro Gly Arg 275 280 285 GAC CGG CGC ACA GAG GAA GAG AATCTC CGC AAG AAA GGG GAG CCT CAC 912 Asp Arg Arg Thr Glu Glu Glu Asn LeuArg Lys Lys Gly Glu Pro His 290 295 300 CAC GAG CTG CCC CCA GGG AGC ACTAAG CGA GCA CTG CCC AAC AAC ACC 960 His Glu Leu Pro Pro Gly Ser Thr LysArg Ala Leu Pro Asn Asn Thr 305 310 315 320 AGC TCC TCT CCC CAG CCA AAGAAG AAA CCA CTG GAT GGA GAA TAT TTC 1008 Ser Ser Ser Pro Gln Pro Lys LysLys Pro Leu Asp Gly Glu Tyr Phe 325 330 335 ACC CTT CAG ATC CGT GGG CGTGAG CGC TTC GAG ATG TTC CGA GAG GAT 1056 Thr Leu Gln Ile Arg Gly Arg GluArg Phe Glu Met Phe Arg Glu Asp 340 345 350 CTG AAG GCC CTC AAG GAG AAGCTG AAG GCC CTG GAG GAG AAG CTG AAG 1104 Leu Lys Ala Leu Lys Glu Lys LeuLys Ala Leu Glu Glu Lys Leu Lys 355 360 365 GCC CTG GAG GAG AAG CTG AAGGCA CTA GTG GGG GAG CGA TGA TGA 1149 Ala Leu Glu Glu Lys Leu Lys Ala LeuVal Gly Glu Arg 370 375 380 381 amino acids amino acid linear proteinnot provided 36 Met Ala Thr Ala Pro Pro Thr Asp Val Ser Leu Gly Asp GluLeu His 1 5 10 15 Leu Asp Gly Glu Asp Val Ala Met Ala His Ala Asp AlaLeu Asp Asp 20 25 30 Phe Asp Leu Asp Met Leu Gly Asp Gly Asp Ser Pro GlyPro Gly Phe 35 40 45 Thr Pro His Asp Ser Ala Pro Tyr Gly Ala Leu Asp MetAla Asp Phe 50 55 60 Glu Phe Glu Gln Met Phe Thr Asp Ala Leu Gly Ile AspGlu Tyr Gly 65 70 75 80 Gly Arg Pro Ala Pro Ala Ala Pro Thr Pro Ala AlaPro Ala Pro Ala 85 90 95 Pro Ser Trp Pro Leu Ser Ser Ser Val Pro Ser GlnLys Thr Tyr Gln 100 105 110 Gly Ser Tyr Gly Phe Arg Leu Gly Phe Leu HisSer Gly Thr Ala Lys 115 120 125 Ser Val Thr Cys Thr Tyr Ser Pro Ala LeuAsn Lys Met Phe Cys Gln 130 135 140 Leu Ala Lys Thr Cys Pro Val Gln LeuTrp Val Asp Ser Thr Pro Pro 145 150 155 160 Pro Gly Thr Arg Val Arg AlaMet Ala Ile Tyr Lys Gln Ser Gln His 165 170 175 Met Thr Glu Val Val ArgArg Cys Pro His His Glu Arg Cys Ser Asp 180 185 190 Ser Asp Gly Leu AlaPro Pro Gln His Leu Ile Arg Val Glu Gly Asn 195 200 205 Leu Arg Val GluTyr Leu Asp Asp Arg Asn Thr Phe Arg His Ser Val 210 215 220 Val Val ProTyr Glu Pro Pro Glu Val Gly Ser Asp Cys Thr Thr Ile 225 230 235 240 HisTyr Asn Tyr Met Cys Asn Ser Ser Cys Met Gly Gly Met Asn Arg 245 250 255Arg Pro Ile Leu Thr Ile Ile Thr Leu Glu Asp Ser Ser Gly Asn Leu 260 265270 Leu Gly Arg Asn Ser Phe Glu Val Arg Val Cys Ala Cys Pro Gly Arg 275280 285 Asp Arg Arg Thr Glu Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His290 295 300 His Glu Leu Pro Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn AsnThr 305 310 315 320 Ser Ser Ser Pro Gln Pro Lys Lys Lys Pro Leu Asp GlyGlu Tyr Phe 325 330 335 Thr Leu Gln Ile Arg Gly Arg Glu Arg Phe Glu MetPhe Arg Glu Asp 340 345 350 Leu Lys Ala Leu Lys Glu Lys Leu Lys Ala LeuGlu Glu Lys Leu Lys 355 360 365 Ala Leu Glu Glu Lys Leu Lys Ala Leu ValGly Glu Arg 370 375 380 1611 base pairs nucleic acid single linear cDNAnot provided CDS 1..1605 37 ATG GCC CAG GTG CAG CTG CAG GAG TCA GGG GCAGAG CTT GTG GGG TCA 48 Met Ala Gln Val Gln Leu Gln Glu Ser Gly Ala GluLeu Val Gly Ser 1 5 10 15 GGG GCC TCA GTC AAG TTG TCC TGC ACA GCT TCTGGC TTC AAC ATT AAA 96 Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser GlyPhe Asn Ile Lys 20 25 30 GAC TAC TAT ATG CAC TGG GTG AAG CAG AGG CCT GAACAG GGC CTG GAG 144 Asp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Glu GlnGly Leu Glu 35 40 45 TGG ATT GGA TGG ATT GAT CCT GAG AAT GGT GAT ACT GAATAT GCC CCG 192 Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu TyrAla Pro 50 55 60 AAG TTC CAG GGC AAG GCC ACT ATG ACT GCA GAC ACA TCC TCCAAT ACA 240 Lys Phe Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser AsnThr 65 70 75 80 GCC TAC CTG CAG CTC AGC AGC CTG GCA TCT GAG GAC ACT GCCGTC TAT 288 Ala Tyr Leu Gln Leu Ser Ser Leu Ala Ser Glu Asp Thr Ala ValTyr 85 90 95 TAT TGT AAT TTT TAC GGG GAT GCT TTG GAC TAC TGG GGC CAA GGGACC 336 Tyr Cys Asn Phe Tyr Gly Asp Ala Leu Asp Tyr Trp Gly Gln Gly Thr100 105 110 ACG GTC ACC GTC TCC TCA GGT GGA GGC GGT TCA GGC GGA GGT GGCTCT 384 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser115 120 125 GGC GGT GGC GGA TCG GAT GTT TTG ATG ACC CAA ACT CCA CTC ACTTTG 432 Gly Gly Gly Gly Ser Asp Val Leu Met Thr Gln Thr Pro Leu Thr Leu130 135 140 TCG GTT ACC ATT GGA CAA CCA GCC TCC ATC TCT TGC AAG TCA AGTCAG 480 Ser Val Thr Ile Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln145 150 155 160 AGC CTC TTG GAT AGT GAT GGA AAG ACA TAT TTG AAT TGG TTGTTA CAG 528 Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu LeuGln 165 170 175 AGG CCA GGC CAG TCT CCA AAG CGC CTA ATC TAT CTG GTG TCTAAA CTG 576 Arg Pro Gly Gln Ser Pro Lys Arg Leu Ile Tyr Leu Val Ser LysLeu 180 185 190 GAC TCT GGA GTC CCT GAC AGG TTC ACT GGC AGT GGA TCA GGGACA GAT 624 Asp Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly ThrAsp 195 200 205 TTC ACA CTG AAA ATC AAC AGA GTG GAG GCT GAG GAT TTG GGAGTT TAT 672 Phe Thr Leu Lys Ile Asn Arg Val Glu Ala Glu Asp Leu Gly ValTyr 210 215 220 TAT TGC TGG CAA GGT ACA CAT TCT CCG CTC ACG TTC GGT GCTGGG ACC 720 Tyr Cys Trp Gln Gly Thr His Ser Pro Leu Thr Phe Gly Ala GlyThr 225 230 235 240 AAG CTG GAG CTG AAA CGG GCG GCC GCA TTG CAG ACG CGTCGA CCT GCA 768 Lys Leu Glu Leu Lys Arg Ala Ala Ala Leu Gln Thr Arg ArgPro Ala 245 250 255 CCA GCA GCT CCT ACA CCG GCG GCC CCT GCA CCA GCC CCCTCC TGG CCC 816 Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro SerTrp Pro 260 265 270 CTG TCA TCT TCT GTC CCT TCC CAG AAA ACC TAC CAG GGCAGC TAC GGT 864 Leu Ser Ser Ser Val Pro Ser Gln Lys Thr Tyr Gln Gly SerTyr Gly 275 280 285 TTC CGT CTG GGC TTC TTG CAT TCT GGG ACA GCC AAG TCTGTG ACT TGC 912 Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys Ser ValThr Cys 290 295 300 ACG TAC TCC CCT GCC CTC AAC AAG ATG TTT TGC CAA CTGGCC AAG ACC 960 Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys Gln Leu AlaLys Thr 305 310 315 320 TGC CCT GTG CAG CTG TGG GTT GAT TCC ACA CCC CCGCCC GGC ACC CGC 1008 Cys Pro Val Gln Leu Trp Val Asp Ser Thr Pro Pro ProGly Thr Arg 325 330 335 GTC CGC GCC ATG GCC ATC TAC AAG CAG TCA CAG CACATG ACG GAG GTT 1056 Val Arg Ala Met Ala Ile Tyr Lys Gln Ser Gln His MetThr Glu Val 340 345 350 GTG AGG CGC TGC CCC CAC CAT GAG CGC TGC TCA GATAGC GAT GGT CTG 1104 Val Arg Arg Cys Pro His His Glu Arg Cys Ser Asp SerAsp Gly Leu 355 360 365 GCC CCT CCT CAG CAT CTT ATC CGA GTG GAA GGA AATTTG CGT GTG GAG 1152 Ala Pro Pro Gln His Leu Ile Arg Val Glu Gly Asn LeuArg Val Glu 370 375 380 TAT TTG GAT GAC AGA AAC ACT TTT CGA CAT AGT GTGGTG GTG CCC TAT 1200 Tyr Leu Asp Asp Arg Asn Thr Phe Arg His Ser Val ValVal Pro Tyr 385 390 395 400 GAG CCG CCT GAG GTT GGC TCT GAC TGT ACC ACCATC CAC TAC AAC TAC 1248 Glu Pro Pro Glu Val Gly Ser Asp Cys Thr Thr IleHis Tyr Asn Tyr 405 410 415 ATG TGT AAC AGT TCC TGC ATG GGC GGC ATG AACCGG AGG CCC ATC CTC 1296 Met Cys Asn Ser Ser Cys Met Gly Gly Met Asn ArgArg Pro Ile Leu 420 425 430 ACC ATC ATC ACA CTG GAA GAC TCC AGT GGT AATCTA CTG GGA CGG AAC 1344 Thr Ile Ile Thr Leu Glu Asp Ser Ser Gly Asn LeuLeu Gly Arg Asn 435 440 445 AGC TTT GAG GTG CGT GTT TGT GCC TGT CCT GGGAGA GAC CGG CGC ACA 1392 Ser Phe Glu Val Arg Val Cys Ala Cys Pro Gly ArgAsp Arg Arg Thr 450 455 460 GAG GAA GAG AAT CTC CGC AAG AAA GGG GAG CCTCAC CAC GAG CTG CCC 1440 Glu Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro HisHis Glu Leu Pro 465 470 475 480 CCA GGG AGC ACT AAG CGA GCA CTG CCC AACAAC ACC AGC TCC TCT CCC 1488 Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn AsnThr Ser Ser Ser Pro 485 490 495 CAG CCA AAG AAG AAA CCA CTG GAT GGG GATCTG AAG GCC CTC AAG GAG 1536 Gln Pro Lys Lys Lys Pro Leu Asp Gly Asp LeuLys Ala Leu Lys Glu 500 505 510 AAG CTG AAG GCC CTG GAG GAG AAG CTG AAGGCC CTG GAG GAG AAG CTG 1584 Lys Leu Lys Ala Leu Glu Glu Lys Leu Lys AlaLeu Glu Glu Lys Leu 515 520 525 AAG GCA CTA GTG GGG GAG CGA TGATGA 1611Lys Ala Leu Val Gly Glu Arg 530 535 535 amino acids amino acid linearprotein not provided 38 Met Ala Gln Val Gln Leu Gln Glu Ser Gly Ala GluLeu Val Gly Ser 1 5 10 15 Gly Ala Ser Val Lys Leu Ser Cys Thr Ala SerGly Phe Asn Ile Lys 20 25 30 Asp Tyr Tyr Met His Trp Val Lys Gln Arg ProGlu Gln Gly Leu Glu 35 40 45 Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly AspThr Glu Tyr Ala Pro 50 55 60 Lys Phe Gln Gly Lys Ala Thr Met Thr Ala AspThr Ser Ser Asn Thr 65 70 75 80 Ala Tyr Leu Gln Leu Ser Ser Leu Ala SerGlu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Asn Phe Tyr Gly Asp Ala Leu AspTyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly GlyGly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Val LeuMet Thr Gln Thr Pro Leu Thr Leu 130 135 140 Ser Val Thr Ile Gly Gln ProAla Ser Ile Ser Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asp SerAsp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln 165 170 175 Arg Pro Gly GlnSer Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu 180 185 190 Asp Ser GlyVal Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 195 200 205 Phe ThrLeu Lys Ile Asn Arg Val Glu Ala Glu Asp Leu Gly Val Tyr 210 215 220 TyrCys Trp Gln Gly Thr His Ser Pro Leu Thr Phe Gly Ala Gly Thr 225 230 235240 Lys Leu Glu Leu Lys Arg Ala Ala Ala Leu Gln Thr Arg Arg Pro Ala 245250 255 Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro260 265 270 Leu Ser Ser Ser Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser TyrGly 275 280 285 Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys Ser ValThr Cys 290 295 300 Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys Gln LeuAla Lys Thr 305 310 315 320 Cys Pro Val Gln Leu Trp Val Asp Ser Thr ProPro Pro Gly Thr Arg 325 330 335 Val Arg Ala Met Ala Ile Tyr Lys Gln SerGln His Met Thr Glu Val 340 345 350 Val Arg Arg Cys Pro His His Glu ArgCys Ser Asp Ser Asp Gly Leu 355 360 365 Ala Pro Pro Gln His Leu Ile ArgVal Glu Gly Asn Leu Arg Val Glu 370 375 380 Tyr Leu Asp Asp Arg Asn ThrPhe Arg His Ser Val Val Val Pro Tyr 385 390 395 400 Glu Pro Pro Glu ValGly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr 405 410 415 Met Cys Asn SerSer Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu 420 425 430 Thr Ile IleThr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn 435 440 445 Ser PheGlu Val Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr 450 455 460 GluGlu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro 465 470 475480 Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro 485490 495 Gln Pro Lys Lys Lys Pro Leu Asp Gly Asp Leu Lys Ala Leu Lys Glu500 505 510 Lys Leu Lys Ala Leu Glu Glu Lys Leu Lys Ala Leu Glu Glu LysLeu 515 520 525 Lys Ala Leu Val Gly Glu Arg 530 535 1065 base pairsnucleic acid single linear cDNA not provided CDS 1..1059 39 ATG GGA GAATAT TTC ACC CTT CAG ATC CGT GGG CGT GAG CGC TTC GAG 48 Met Gly Glu TyrPhe Thr Leu Gln Ile Arg Gly Arg Glu Arg Phe Glu 1 5 10 15 ATG TTC CGAGAG CTG AAT GAG GCC TTG GAA CTC AAG GAT GCC CAG GCT 96 Met Phe Arg GluLeu Asn Glu Ala Leu Glu Leu Lys Asp Ala Gln Ala 20 25 30 GGG AAG GAG CCAGGG GGG AGC AGG GCT CAC TCC AGC CAC CTG AAG TCC 144 Gly Lys Glu Pro GlyGly Ser Arg Ala His Ser Ser His Leu Lys Ser 35 40 45 AAA AAG GGT CAG TCTACC TCC CGC CAT AAA AAA CTC ATG TTC AAG ACA 192 Lys Lys Gly Gln Ser ThrSer Arg His Lys Lys Leu Met Phe Lys Thr 50 55 60 GAA GGG CCT GAC TCA GACGGT CGA CCT GCA CCA GCA GCT CCT ACA CCG 240 Glu Gly Pro Asp Ser Asp GlyArg Pro Ala Pro Ala Ala Pro Thr Pro 65 70 75 80 GCG GCC CCT GCA CCA GCCCCC TCC TGG CCC CTG TCA TCT TCT GTC CCT 288 Ala Ala Pro Ala Pro Ala ProSer Trp Pro Leu Ser Ser Ser Val Pro 85 90 95 TCC CAG AAA ACC TAC CAG GGCAGC TAC GGT TTC CGT CTG GGC TTC TTG 336 Ser Gln Lys Thr Tyr Gln Gly SerTyr Gly Phe Arg Leu Gly Phe Leu 100 105 110 CAT TCT GGG ACA GCC AAG TCTGTG ACT TGC ACG TAC TCC CCT GCC CTC 384 His Ser Gly Thr Ala Lys Ser ValThr Cys Thr Tyr Ser Pro Ala Leu 115 120 125 AAC AAG ATG TTT TGC CAA CTGGCC AAG ACC TGC CCT GTG CAG CTG TGG 432 Asn Lys Met Phe Cys Gln Leu AlaLys Thr Cys Pro Val Gln Leu Trp 130 135 140 GTT GAT TCC ACA CCC CCG CCCGGC ACC CGC GTC CGC GCC ATG GCC ATC 480 Val Asp Ser Thr Pro Pro Pro GlyThr Arg Val Arg Ala Met Ala Ile 145 150 155 160 TAC AAG CAG TCA CAG CACATG ACG GAG GTT GTG AGG CGC TGC CCC CAC 528 Tyr Lys Gln Ser Gln His MetThr Glu Val Val Arg Arg Cys Pro His 165 170 175 CAT GAG CGC TGC TCA GATAGC GAT GGT CTG GCC CCT CCT CAG CAT CTT 576 His Glu Arg Cys Ser Asp SerAsp Gly Leu Ala Pro Pro Gln His Leu 180 185 190 ATC CGA GTG GAA GGA AATTTG CGT GTG GAG TAT TTG GAT GAC AGA AAC 624 Ile Arg Val Glu Gly Asn LeuArg Val Glu Tyr Leu Asp Asp Arg Asn 195 200 205 ACT TTT CGA CAT AGT GTGGTG GTG CCC TAT GAG CCG CCT GAG GTT GGC 672 Thr Phe Arg His Ser Val ValVal Pro Tyr Glu Pro Pro Glu Val Gly 210 215 220 TCT GAC TGT ACC ACC ATCCAC TAC AAC TAC ATG TGT AAC AGT TCC TGC 720 Ser Asp Cys Thr Thr Ile HisTyr Asn Tyr Met Cys Asn Ser Ser Cys 225 230 235 240 ATG GGC GGC ATG AACCGG AGG CCC ATC CTC ACC ATC ATC ACA CTG GAA 768 Met Gly Gly Met Asn ArgArg Pro Ile Leu Thr Ile Ile Thr Leu Glu 245 250 255 GAC TCC AGT GGT AATCTA CTG GGA CGG AAC AGC TTT GAG GTG CGT GTT 816 Asp Ser Ser Gly Asn LeuLeu Gly Arg Asn Ser Phe Glu Val Arg Val 260 265 270 TGT GCC TGT CCT GGGAGA GAC CGG CGC ACA GAG GAA GAG AAT CTC CGC 864 Cys Ala Cys Pro Gly ArgAsp Arg Arg Thr Glu Glu Glu Asn Leu Arg 275 280 285 AAG AAA GGG GAG CCTCAC CAC GAG CTG CCC CCA GGG AGC ACT AAG CGA 912 Lys Lys Gly Glu Pro HisHis Glu Leu Pro Pro Gly Ser Thr Lys Arg 290 295 300 GCA CTG CCC AAC AACACC AGC TCC TCT CCC CAG CCA AAG AAG AAA CCA 960 Ala Leu Pro Asn Asn ThrSer Ser Ser Pro Gln Pro Lys Lys Lys Pro 305 310 315 320 CTG GAT GGG GATCTG AAG GCC CTC AAG GAG AAG CTG AAG GCC CTG GAG 1008 Leu Asp Gly Asp LeuLys Ala Leu Lys Glu Lys Leu Lys Ala Leu Glu 325 330 335 GAG AAG CTG AAGGCC CTG GAG GAG AAG CTG AAG GCA CTA GTG GGG GAG 1056 Glu Lys Leu Lys AlaLeu Glu Glu Lys Leu Lys Ala Leu Val Gly Glu 340 345 350 CGA TGATGA 1065Arg 353 amino acids amino acid linear protein not provided 40 Met GlyGlu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu Arg Phe Glu 1 5 10 15 MetPhe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp Ala Gln Ala 20 25 30 GlyLys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His Leu Lys Ser 35 40 45 LysLys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr 50 55 60 GluGly Pro Asp Ser Asp Gly Arg Pro Ala Pro Ala Ala Pro Thr Pro 65 70 75 80Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser Val Pro 85 90 95Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly Phe Leu 100 105110 His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu 115120 125 Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln Leu Trp130 135 140 Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met AlaIle 145 150 155 160 Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg ArgCys Pro His 165 170 175 His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala ProPro Gln His Leu 180 185 190 Ile Arg Val Glu Gly Asn Leu Arg Val Glu TyrLeu Asp Asp Arg Asn 195 200 205 Thr Phe Arg His Ser Val Val Val Pro TyrGlu Pro Pro Glu Val Gly 210 215 220 Ser Asp Cys Thr Thr Ile His Tyr AsnTyr Met Cys Asn Ser Ser Cys 225 230 235 240 Met Gly Gly Met Asn Arg ArgPro Ile Leu Thr Ile Ile Thr Leu Glu 245 250 255 Asp Ser Ser Gly Asn LeuLeu Gly Arg Asn Ser Phe Glu Val Arg Val 260 265 270 Cys Ala Cys Pro GlyArg Asp Arg Arg Thr Glu Glu Glu Asn Leu Arg 275 280 285 Lys Lys Gly GluPro His His Glu Leu Pro Pro Gly Ser Thr Lys Arg 290 295 300 Ala Leu ProAsn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys Lys Pro 305 310 315 320 LeuAsp Gly Asp Leu Lys Ala Leu Lys Glu Lys Leu Lys Ala Leu Glu 325 330 335Glu Lys Leu Lys Ala Leu Glu Glu Lys Leu Lys Ala Leu Val Gly Glu 340 345350 Arg 963 base pairs nucleic acid single linear cDNA not provided CDS1..957 41 ATG GGA GAA TAT TTC ACC CTT CAG ATC CGT GGG CGT GAG CGC TTCGAG 48 Met Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu Arg Phe Glu 15 10 15 ATG TTC CGA GAG CTG AAT GAG GCC TTG GAA CTC AAG GAT GCC CAG GCT96 Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp Ala Gln Ala 20 2530 GGG AAG GAG CCA GGT CGA CCT GCA CCA GCA GCT CCT ACA CCG GCG GCC 144Gly Lys Glu Pro Gly Arg Pro Ala Pro Ala Ala Pro Thr Pro Ala Ala 35 40 45CCT GCA CCA GCC CCC TCC TGG CCC CTG TCA TCT TCT GTC CCT TCC CAG 192 ProAla Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser Val Pro Ser Gln 50 55 60 AAAACC TAC CAG GGC AGC TAC GGT TTC CGT CTG GGC TTC TTG CAT TCT 240 Lys ThrTyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser 65 70 75 80 GGGACA GCC AAG TCT GTG ACT TGC ACG TAC TCC CCT GCC CTC AAC AAG 288 Gly ThrAla Lys Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys 85 90 95 ATG TTTTGC CAA CTG GCC AAG ACC TGC CCT GTG CAG CTG TGG GTT GAT 336 Met Phe CysGln Leu Ala Lys Thr Cys Pro Val Gln Leu Trp Val Asp 100 105 110 TCC ACACCC CCG CCC GGC ACC CGC GTC CGC GCC ATG GCC ATC TAC AAG 384 Ser Thr ProPro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys 115 120 125 CAG TCACAG CAC ATG ACG GAG GTT GTG AGG CGC TGC CCC CAC CAT GAG 432 Gln Ser GlnHis Met Thr Glu Val Val Arg Arg Cys Pro His His Glu 130 135 140 CGC TGCTCA GAT AGC GAT GGT CTG GCC CCT CCT CAG CAT CTT ATC CGA 480 Arg Cys SerAsp Ser Asp Gly Leu Ala Pro Pro Gln His Leu Ile Arg 145 150 155 160 GTGGAA GGA AAT TTG CGT GTG GAG TAT TTG GAT GAC AGA AAC ACT TTT 528 Val GluGly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn Thr Phe 165 170 175 CGACAT AGT GTG GTG GTG CCC TAT GAG CCG CCT GAG GTT GGC TCT GAC 576 Arg HisSer Val Val Val Pro Tyr Glu Pro Pro Glu Val Gly Ser Asp 180 185 190 TGTACC ACC ATC CAC TAC AAC TAC ATG TGT AAC AGT TCC TGC ATG GGC 624 Cys ThrThr Ile His Tyr Asn Tyr Met Cys Asn Ser Ser Cys Met Gly 195 200 205 GGCATG AAC CGG AGG CCC ATC CTC ACC ATC ATC ACA CTG GAA GAC TCC 672 Gly MetAsn Arg Arg Pro Ile Leu Thr Ile Ile Thr Leu Glu Asp Ser 210 215 220 AGTGGT AAT CTA CTG GGA CGG AAC AGC TTT GAG GTG CGT GTT TGT GCC 720 Ser GlyAsn Leu Leu Gly Arg Asn Ser Phe Glu Val Arg Val Cys Ala 225 230 235 240TGT CCT GGG AGA GAC CGG CGC ACA GAG GAA GAG AAT CTC CGC AAG AAA 768 CysPro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn Leu Arg Lys Lys 245 250 255GGG GAG CCT CAC CAC GAG CTG CCC CCA GGG AGC ACT AAG CGA GCA CTG 816 GlyGlu Pro His His Glu Leu Pro Pro Gly Ser Thr Lys Arg Ala Leu 260 265 270CCC AAC AAC ACC AGC TCC TCT CCC CAG CCA AAG AAG AAA CCA CTG GAT 864 ProAsn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys Lys Pro Leu Asp 275 280 285GGG GAT CTG AAG GCC CTC AAG GAG AAG CTG AAG GCC CTG GAG GAG AAG 912 GlyAsp Leu Lys Ala Leu Lys Glu Lys Leu Lys Ala Leu Glu Glu Lys 290 295 300CTG AAG GCC CTG GAG GAG AAG CTG AAG GCA CTA GTG GGG GAG CGA 957 Leu LysAla Leu Glu Glu Lys Leu Lys Ala Leu Val Gly Glu Arg 305 310 315 TGATGA963 319 amino acids amino acid linear protein not provided 42 Met GlyGlu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu Arg Phe Glu 1 5 10 15 MetPhe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp Ala Gln Ala 20 25 30 GlyLys Glu Pro Gly Arg Pro Ala Pro Ala Ala Pro Thr Pro Ala Ala 35 40 45 ProAla Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser Val Pro Ser Gln 50 55 60 LysThr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser 65 70 75 80Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys 85 90 95Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln Leu Trp Val Asp 100 105110 Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys 115120 125 Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys Pro His His Glu130 135 140 Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln His Leu IleArg 145 150 155 160 Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp ArgAsn Thr Phe 165 170 175 Arg His Ser Val Val Val Pro Tyr Glu Pro Pro GluVal Gly Ser Asp 180 185 190 Cys Thr Thr Ile His Tyr Asn Tyr Met Cys AsnSer Ser Cys Met Gly 195 200 205 Gly Met Asn Arg Arg Pro Ile Leu Thr IleIle Thr Leu Glu Asp Ser 210 215 220 Ser Gly Asn Leu Leu Gly Arg Asn SerPhe Glu Val Arg Val Cys Ala 225 230 235 240 Cys Pro Gly Arg Asp Arg ArgThr Glu Glu Glu Asn Leu Arg Lys Lys 245 250 255 Gly Glu Pro His His GluLeu Pro Pro Gly Ser Thr Lys Arg Ala Leu 260 265 270 Pro Asn Asn Thr SerSer Ser Pro Gln Pro Lys Lys Lys Pro Leu Asp 275 280 285 Gly Asp Leu LysAla Leu Lys Glu Lys Leu Lys Ala Leu Glu Glu Lys 290 295 300 Leu Lys AlaLeu Glu Glu Lys Leu Lys Ala Leu Val Gly Glu Arg 305 310 315 1011 basepairs nucleic acid single linear cDNA not provided CDS 1..1005 43 ATGGGA GAA TAT TTC ACC CTT CAG ATC CGT GGG CGT GAG CGC TTC GAG 48 Met GlyGlu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu Arg Phe Glu 1 5 10 15 ATGTTC CGA GAG CTG AAT GAG GCC TTG GAA CTC AAG GAT GCC CAG GCT 96 Met PheArg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp Ala Gln Ala 20 25 30 GGG AAGGAG CCA GGT CGA GGA GGT GGT GGC TCT GGA GGC GGA GGA TCC 144 Gly Lys GluPro Gly Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 35 40 45 GGC GGT GGAGGT TCT CGA CCT GCA CCA GCA GCT CCT ACA CCG GCG GCC 192 Gly Gly Gly GlySer Arg Pro Ala Pro Ala Ala Pro Thr Pro Ala Ala 50 55 60 CCT GCA CCA GCCCCC TCC TGG CCC CTG TCA TCT TCT GTC CCT TCC CAG 240 Pro Ala Pro Ala ProSer Trp Pro Leu Ser Ser Ser Val Pro Ser Gln 65 70 75 80 AAA ACC TAC CAGGGC AGC TAC GGT TTC CGT CTG GGC TTC TTG CAT TCT 288 Lys Thr Tyr Gln GlySer Tyr Gly Phe Arg Leu Gly Phe Leu His Ser 85 90 95 GGG ACA GCC AAG TCTGTG ACT TGC ACG TAC TCC CCT GCC CTC AAC AAG 336 Gly Thr Ala Lys Ser ValThr Cys Thr Tyr Ser Pro Ala Leu Asn Lys 100 105 110 ATG TTT TGC CAA CTGGCC AAG ACC TGC CCT GTG CAG CTG TGG GTT GAT 384 Met Phe Cys Gln Leu AlaLys Thr Cys Pro Val Gln Leu Trp Val Asp 115 120 125 TCC ACA CCC CCG CCCGGC ACC CGC GTC CGC GCC ATG GCC ATC TAC AAG 432 Ser Thr Pro Pro Pro GlyThr Arg Val Arg Ala Met Ala Ile Tyr Lys 130 135 140 CAG TCA CAG CAC ATGACG GAG GTT GTG AGG CGC TGC CCC CAC CAT GAG 480 Gln Ser Gln His Met ThrGlu Val Val Arg Arg Cys Pro His His Glu 145 150 155 160 CGC TGC TCA GATAGC GAT GGT CTG GCC CCT CCT CAG CAT CTT ATC CGA 528 Arg Cys Ser Asp SerAsp Gly Leu Ala Pro Pro Gln His Leu Ile Arg 165 170 175 GTG GAA GGA AATTTG CGT GTG GAG TAT TTG GAT GAC AGA AAC ACT TTT 576 Val Glu Gly Asn LeuArg Val Glu Tyr Leu Asp Asp Arg Asn Thr Phe 180 185 190 CGA CAT AGT GTGGTG GTG CCC TAT GAG CCG CCT GAG GTT GGC TCT GAC 624 Arg His Ser Val ValVal Pro Tyr Glu Pro Pro Glu Val Gly Ser Asp 195 200 205 TGT ACC ACC ATCCAC TAC AAC TAC ATG TGT AAC AGT TCC TGC ATG GGC 672 Cys Thr Thr Ile HisTyr Asn Tyr Met Cys Asn Ser Ser Cys Met Gly 210 215 220 GGC ATG AAC CGGAGG CCC ATC CTC ACC ATC ATC ACA CTG GAA GAC TCC 720 Gly Met Asn Arg ArgPro Ile Leu Thr Ile Ile Thr Leu Glu Asp Ser 225 230 235 240 AGT GGT AATCTA CTG GGA CGG AAC AGC TTT GAG GTG CGT GTT TGT GCC 768 Ser Gly Asn LeuLeu Gly Arg Asn Ser Phe Glu Val Arg Val Cys Ala 245 250 255 TGT CCT GGGAGA GAC CGG CGC ACA GAG GAA GAG AAT CTC CGC AAG AAA 816 Cys Pro Gly ArgAsp Arg Arg Thr Glu Glu Glu Asn Leu Arg Lys Lys 260 265 270 GGG GAG CCTCAC CAC GAG CTG CCC CCA GGG AGC ACT AAG CGA GCA CTG 864 Gly Glu Pro HisHis Glu Leu Pro Pro Gly Ser Thr Lys Arg Ala Leu 275 280 285 CCC AAC AACACC AGC TCC TCT CCC CAG CCA AAG AAG AAA CCA CTG GAT 912 Pro Asn Asn ThrSer Ser Ser Pro Gln Pro Lys Lys Lys Pro Leu Asp 290 295 300 GGG GAT CTGAAG GCC CTC AAG GAG AAG CTG AAG GCC CTG GAG GAG AAG 960 Gly Asp Leu LysAla Leu Lys Glu Lys Leu Lys Ala Leu Glu Glu Lys 305 310 315 320 CTG AAGGCC CTG GAG GAG AAG CTG AAG GCA CTA GTG GGG GAG CGA 1005 Leu Lys Ala LeuGlu Glu Lys Leu Lys Ala Leu Val Gly Glu Arg 325 330 335 TGATGA 1011 335amino acids amino acid linear protein not provided 44 Met Gly Glu TyrPhe Thr Leu Gln Ile Arg Gly Arg Glu Arg Phe Glu 1 5 10 15 Met Phe ArgGlu Leu Asn Glu Ala Leu Glu Leu Lys Asp Ala Gln Ala 20 25 30 Gly Lys GluPro Gly Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 35 40 45 Gly Gly GlyGly Ser Arg Pro Ala Pro Ala Ala Pro Thr Pro Ala Ala 50 55 60 Pro Ala ProAla Pro Ser Trp Pro Leu Ser Ser Ser Val Pro Ser Gln 65 70 75 80 Lys ThrTyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser 85 90 95 Gly ThrAla Lys Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys 100 105 110 MetPhe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln Leu Trp Val Asp 115 120 125Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys 130 135140 Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys Pro His His Glu 145150 155 160 Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln His Leu IleArg 165 170 175 Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg AsnThr Phe 180 185 190 Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu ValGly Ser Asp 195 200 205 Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn SerSer Cys Met Gly 210 215 220 Gly Met Asn Arg Arg Pro Ile Leu Thr Ile IleThr Leu Glu Asp Ser 225 230 235 240 Ser Gly Asn Leu Leu Gly Arg Asn SerPhe Glu Val Arg Val Cys Ala 245 250 255 Cys Pro Gly Arg Asp Arg Arg ThrGlu Glu Glu Asn Leu Arg Lys Lys 260 265 270 Gly Glu Pro His His Glu LeuPro Pro Gly Ser Thr Lys Arg Ala Leu 275 280 285 Pro Asn Asn Thr Ser SerSer Pro Gln Pro Lys Lys Lys Pro Leu Asp 290 295 300 Gly Asp Leu Lys AlaLeu Lys Glu Lys Leu Lys Ala Leu Glu Glu Lys 305 310 315 320 Leu Lys AlaLeu Glu Glu Lys Leu Lys Ala Leu Val Gly Glu Arg 325 330 335 24 basepairs nucleic acid single linear cDNA not provided 45 CGGATCCTCTCGGAACATCT CGAA 24 749 base pairs nucleic acid single linear cDNA notprovided 46 GCCATGGCCC AGGTGCAGCT GCAGGAGTCA GGGGCAGAGC TTGTGGGGTCAGGGGCCTCA 60 GTCAAGTTGT CCTGCACAGC TTCTGGCTTC AACATTAAAG ACTACTATATGCACTGGGTG 120 AAGCAGAGGC CTGAACAGGG CCTGGAGTGG ATTGGATGGA TTGATCCTGAGAATGGTGAT 180 ACTGAATATG CCCCGAAGTT CCAGGGCAAG GCCACTATGA CTGCAGACACATCCTCCAAT 240 ACAGCCTACC TGCAGCTCAG CAGCCTGGCA TCTGAGGACA CTGCCGTCTATTATTGTAAT 300 TTTTACGGGG ATGCTTTGGA CTACTGGGGC CAAGGGACCA CGGTCACCGTCTCCTCAGGT 360 GGAGGCGGTT CAGGCGGAGG TGGCTCTGGC GGTGGCGGAT CGGATGTTTTGATGACCCAA 420 ACTCCACTCA CTTTGTCGGT TACCATTGGA CAACCAGCCT CCATCTCTTGCAAGTCAAGT 480 CAGAGCCTCT TGGATAGTGA TGGAAAGACA TATTTGAATT GGTTGTTACAGAGGCCAGGC 540 CAGTCTCCAA AGCGCCTAAT CTATCTGGTG TCTAAACTGG ACTCTGGAGTCCCTGACAGG 600 TTCACTGGCA GTGGATCAGG GACAGATTTC ACACTGAAAA TCAACAGAGTGGAGGCTGAG 660 GATTTGGGAG TTTATTATTG CTGGCAAGGT ACACATTCTC CGCTCACGTTCGGTGCTGGG 720 ACCAAGCTGG AGCTGAAACG GGCGGCCGC 749 45 base pairs nucleicacid single linear cDNA not provided 47 AAGCTTGAAT TCGTTAACGC CACCATGGGAGAATATTTCA CCCTT 45 24 base pairs nucleic acid single linear cDNA notprovided 48 GGGTCGACCT GGCTCCTTCC CAGC 24 141 base pairs nucleic acidsingle linear other nucleic acid /desc = “oligonucleotide” not provided49 AAGCTTGAAT TCGTTAACGC CACCATGGGA GAATATTTCA CCCTTCAGAT CCGTGGGCGT 60GAGCGCTTCG AGATGTTCCG AGAGCTGAAT GAGGCCTTGG AACTCAAGGA TGCCCAGGC1 20GGGAAGGAGC CAGGTCGACC C 141 27 base pairs nucleic acid single linearcDNA not provided 50 GGGTCGACCG TCTGAGTCAG GCCCTTC 27 243 base pairsnucleic acid single linear other nucleic acid /desc = “oligonucleotide”not provided 51 AAGCTTGAAT TCGTTAACGC CACCATGGGA GAATATTTCA CCCTTCAGATCCGTGGGCGT 60 GAGCGCTTCG AGATGTTCCG AGAGCTGAAT GAGGCCTTGG AACTCAAGGATGCCCAGGCT 120 GGGAAGGAGC CAGGGGGGAG CAGGGCTCAC TCCAGCCACC TGAAGTCCAAAAAGGGTCAG 180 TCTACCTCCC GCCATAAAAA ACTCATGTTC AAGACAGAAG GGCCTGACTCAGACGGTCGA 240 CCC 243 48 base pairs nucleic acid single linear cDNA notprovided 52 TCGAGGAGGT GGTGGCTCTG GAGGCGGAGG ATCCGGCGGT GGAGGTTC 48 48base pairs nucleic acid single linear cDNA not provided 53 TCGAGAACCCCTACCGCCGG ATCCTCCGCC TCCAGAGCCA CCACCTCC 48 16 amino acids amino acid<Unknown> linear peptide not provided 54 Gly Gly Gly Ser Gly Gly Gly SerGly Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 26 base pairs nucleic acidsingle linear cDNA not provided 55 GATCCGAACA TGTCCCAACA TGTTGA 26 26base pairs nucleic acid single linear cDNA not provided 56 AGCTTCAACATGTTGGGACA TGGTCG 26 31 amino acids amino acid <Unknown> linear proteinnot provided 57 Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser ThrSer Arg 1 5 10 15 His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro Asp SerAsp Xaa 20 25 30 23 amino acids amino acid <Unknown> linear protein notprovided 58 Ala His Ser Ser Leu Gln Pro Arg Ala Phe Gln Ala Leu Met LysGlu 1 5 10 15 Glu Ser Pro Asn Cys Xaa Xaa 20 6 amino acids amino acid<Unknown> linear peptide not provided 59 Ala His Ser Ser Xaa Xaa 1 5

What is claimed is:
 1. A variant of p53 protein wherein a C-terminalportion of the protein comprising a regulation domain and a part of anoligomerization domain is deleted from residue 326 or from residue 337and replaced by an artificial leucine zipper comprising residues 334-363of SEQ ID No: 26; and a transactivation domain is deleted and replacedby a VP16 transactivation domain.
 2. The variant according to claim 1,wherein an arginine residue at position 182 of p53 is replaced by ahistidine residue.
 3. The variant according to claim 1, wherein residues1 to 74 are deleted.
 4. The variant according to claim 2, whereinresidues 1 to 74 are deleted.
 5. The variant according to claim 1,wherein the VP16 transactivation domain comprises residues 1-83 of SEQID No:
 26. 6. The variant according to claim 2, wherein the VP16transactivation domain comprises residues 1-83 of SEQ ID No:
 26. 7. Acompound having the sequence of SEQ ID No:
 25. 8. The compound accordingto claim 7, wherein a histidine residue is present at position
 182. 9. Acompound having the sequence of SEQ ID No:
 26. 10. The compoundaccording to claim 9, wherein a histidine residue is present at position182.
 11. A composition comprising a variant according to claim 1 and apharmaceutically-acceptable carrier.